Prolonged anesthesia in joints and body spaces

ABSTRACT

Sustained release local anesthetic formulations are administered intra articularly and/or into body spaces/cavities. The formulation is preferably a plurality of injectable microparticles including a local anesthetic and an effective amount of a biocompatible, biodegradable, sustained release material prolonging the release of the local anesthetic and optionally and a pharmaceutically acceptable, i.e., non-toxic, augmenting agent effective to prolong the duration of the local anesthesia for a time period longer than that obtainable without the augmenting agent.

This application is a continuation of U.S. patent application Ser. No.09/109,324, filed Jul. 2, 1998, now U.S. Pat. No. 6,248,345, whichclaims priority from U.S. Provisional Application Serial No. 60/051,601filed Jul. 2, 1997.

FIELD OF THE INVENTION

The present invention is related to sustained release formulations forthe administration of locally active agents and/or diagnostic agents insustained release form intra articularly or in other body spaces. Inparticular embodiments, the invention provides methods and compositionsfor the administration of local anesthetics as well as compositions andmethods for augmenting the duration and potency of local anesthesia, inpatients in need thereof, and for obtaining additional beneficialeffects, in intra articular locations and in all human or animal bodyspaces.

BACKGROUND OF THE INVENTION

Symptomatic treatment of joint pain has hereto been based on the use ofsystemic treatment with steroidal and nonsteroidal antiinflammatoryagents and analgesics as well as localized injection of steroidalantiinflammatories, e.g., intra articular injection, and localanesthetics, either intra articular or proximal to the innervation ofthe painful joint. Localized treatment is generally preferred oversystemic treatment, particularly when treating severe, localized jointpain, in order to avoid the untoward systemic effects associated withthe high levels of both steroidal and nonsteroidal antiinflammatoryagents otherwise required. Local anesthetics alone have previously beeninjected into joint spaces to relieve pain, with mixed results.

While compounds utilized as general anesthetics reduce pain by producinga loss of consciousness, local anesthetics act by producing a loss ofsensation in the localized area of administration in the body. Themechanism by which local anesthetics induce their effect, while nothaving been determined definitively, is generally thought to be basedupon the ability to interfere with the initiation and transmission ofthe nerve impulse. The duration of action of a local anesthetic isproportional to the time during which it is in actual contact with thenervous tissues. Consequently, procedures or formulations that maintainlocalization of the drug at the nerve greatly prolong anesthesia.

Local anesthetics are potentially toxic, yet must remain at the sitelong enough to allow sufficient time for the localized pain to subside.Therefore, it is of great importance that factors such as the choice ofdrug, concentration of drug, and rate and site of administration of drugbe taken into consideration when contemplating their use.

Different devices and formulations are known in the art foradministration of local anesthetics. For example, local anesthetics canbe delivered in solution or suspension by means of injection, infusion,infiltration, irrigation, topically and the like. Injection or infusioncan be carried out acutely, or if prolonged local effects are desired,localized anesthetic agents can be administered continuously by means ofa gravity drip or infusion pump. Thus, local anesthetics such asbupivacaine have been administered by continuous infusion, e.g., forprolonged epidural or intrathecal administration.

Sustained release carriers for local anesthetics have been described.For example, U.S. Pat. Nos. 4,725,442 and 4,622,219 (Haynes) aredirected to methoxyflurane-containing microdroplets coated with aphospholipid prepared by sonication, which are suitable for intradermalor intravenous injection into a patient for inducing local anesthesia.Such microdroplets are said to cause long-term local anesthesia wheninjected intradermally, giving a duration of anesthesia considerablylonger than the longest acting conventional local anesthetic(bupivacaine).

U.S. Pat. No. 5,188,837 (Domb) relates to a microsuspension systemcontaining lipospheres having a layer of a phospholipid imbedded ontheir surface. The core of the liposphere is a solid substance to bedelivered, or the substance to be delivered is dispersed in an inertvehicle. The substance to be delivered can be, e.g., nonsteroidalanti-inflammatory compounds, local anesthetics, water insolublechemotherapeutic agents and steroids.

Other formulations directed to injectable microcapsules, etc. are known.For example, U.S. Pat. No. 5,061,492 describes prolonged releasemicrocapsules of a water-soluble drug in a biodegradable polymer matrixwhich is composed of a copolymer of glycolic acid and a lactic acid. Themicrocapsules are prepared as an injectable preparation in apharmaceutically acceptable vehicle. The particles of water soluble drugare retained in a drug-retaining substance dispersed in a matrix of thelactic/glycolic acid copolymer in a ratio of 100/1 to 50/50 and anaverage molecular weight of 5,000-200,000. The injectable preparation ismade by preparing a water-in-oil emulsion of an aqueous layer of drugand drug retaining substance and an oil layer of the polymer, thickeningand then water-drying.

U.S. Pat. No. 4,938,763 (Dunn, et al.) is related to a biodegradablepolymer for use in providing syringe able, in-situ forming, solidbiodegradable implants for animals. In one aspect of this reference, athermosetting system is utilized which utilizes copolymers which may bederived from polylactides and/or polyglycolides, combinations andmixtures of these and other polymers.

U.S. Pat. No. 4,293,539 (Ludwig, et al.) is directed to controlledrelease formulations comprised of a microbial agent dispersed throughouta copolymer derived from lactic acid and glycolic acid. The copolymer isderived from 60-95% lactic acid and 40-5% glycolic acid by weight, andhas a molecular weight of 6,000-35,000. An effective amount of thecopolymeric formulation is administered by subcutaneous or intramuscularadministration.

WO 94/05265 describes improved biodegradable sustained release systemsconsisting of a polymeric matrix incorporating a local anesthetic forthe prolonged administration of the local anesthetic agent. The devicesare selected on the basis of their degradation profiles: release of thetopical anesthetic in a linear, controlled manner over the period ofpreferably two weeks and degradation in vivo with a half-life of lessthan six months, more preferably two weeks, to avoid localizedinflammation. The disclosure states that an anti-inflammatory can beincorporated into the polymer with the local anesthetic to reduceencapsulation for optimal access of drug to its site of action. Theanti-inflammatories that are said to be useful include steroids such asdexamethasone, cortisone, prednisone, and others routinely administeredorally or by injection.

Several non-glucocorticoids have been reported to prolong the action oflocal anesthetics. Epinephrine in immediate release form is known bythose of ordinary skill in the art to briefly prolong the action ofimmediate release local anesthetics by inducing vasoconstrictionadjacent to the site of injection. However, the duration of prolongationprovided by immediate release epinephrine is on the order of about anhour, at best, in a highly vascularized tissue. This strategy is alsoseverely limited by the risk of gangrene due to prolonged impairment ofblood flow to local tissues. Dextrans and alkalinizing agents have alsobeen suggested as local anesthesia prolonging agents, but haveheretofore been reported to be ineffective for this purpose (Bonica etal., 1990, “Regional Analgesia With Local Anesthetics” THE MANAGEMENT OFPAIN, Second Edition, Volume II, Published, Lea & Febiger, Chapter 94,pages 1890-1892).

Colchicine has been shown to suppress injury-induced ectopic nervedischarge in a model system of chronic pain utilizing injured nerve(Wall et al.), 1995, Textbook of Pain, Third Edition, Publ., ChurchillLivingston, pages 94-98; Devol et al., 1991, A Group Report: Mechanismsof neuropathic pain following peripheral injury. In: Basbaume A I, et al(eds). TOWARDS A NEW PHARMACOTHERAPY OF PAIN, Dahlem Konferenzen, Wiley,Chichester pp. 417-440; Devor et al., 1985, Pain, 22:127-137 at 128; andDevor, 1983, Pain 16:73-86). It has been reported in one study thatcolchicine was given for the treatment of low-back pain, although oralcolchicine has been shown to be ineffective for the same indication(Schnebel et al., 1988, Spine 13(3):354-7). However, it has notheretofore been known to use colchicine to prolong local anesthesia.

A relatively long-acting local anesthetic, bupivacaine hydrochloride, iscommercially available as Marcaine® Hydrochloride in sterile isotonicsolutions with and without epinephrine (as bitartrate) 1:200,000 forinjection via local infiltration, peripheral nerve block, and caudal andlumbar epidural blocks. After injection of Marcaine for caudal, epiduralor peripheral nerve block in man, peak levels of bupivacaine in theblood are reached in 30 to 45 minutes, followed by a decline toinsignificant levels during the next three to six hours.

In addition, polymer microparticles have long been used for both medicaland non-medical applications where sustained release of an agent ofinterest is desired. Nevertheless, prior to the present invention, itwould have been expected that polymer microparticles in a joint spacewould scratch the extremely smooth and slippery opposed intra articularsurfaces or otherwise irritate or inflame the joint. Thus, the need foran effective method and formulation for delivering pain relief and otherpharmaceutical or diagnostic treatments to the intra articular space hasremained unmet until the present invention. Further, the need for aneffective method and formulation for delivering pain relief and otherpharmaceutical or diagnostic treatments to all body spaces has remainedunmet until the present invention.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide a biodegradablesustained release dosage form for providing prolonged administration ofan active agent for the treatment and/or diagnosis of joint pain and/orother intra articular conditions in humans and animals. Moreparticularly, it is an object of the invention to provide a localanesthetic in a biocompatible, biodegradable sustained release form incombination with an amount of an augmenting agent effective to enhanceand prolong local anesthesia in joints and body spaces/cavities.

It is a further object of the present invention to provide a method forprolonging the effect of a local anesthetic agent in joints and/or bodyspaces/cavities and to further provide a prolonged and beneficialantiinflammatory effect.

It is a further object of the present invention to provide abiocompatible, biodegradable controlled release dosage form forproviding prolonged local anesthetic treatment of body spaces in humansand animals, with or without other active agents described herein.

In accordance with the above-mentioned objects and others, the inventionis related to formulations and methods for the localized and prolongedintra articular administration of active agents by the intra articularadministration of a sustained release formulation according to theinvention.

The sustained release formulation preferably comprises any agentsuitable for the treatment or diagnosis of an intra articular condition.

The method according to the invention includes, for example,administering into an articular joint, a formulation of a biocompatiblesustained release material and one or more active agents suitable forthe purpose. The active agents can include one or more enzymes,anti-infectives, antibodies, and the like, diagnostic agents, as well aslocal anesthetics, local anesthesia augmenting agents and combinationsthereof.

The active agents are preferably a local anesthetic and a non-toxicaugmenting agent effective to potentiate or prolong the action of thelocal anesthetic effect. The sustained release material is in the form,e.g., of a plurality of microparticles including local anesthetic andsaid microparticles are suspended in a pharmaceutically acceptablevehicle for injection.

The formulation can include a local anesthetic augmenting agent, atleast a portion of which is optionally incorporated in the sustainedrelease material. In addition, at least a portion of the augmentingagent may optionally be in immediate release form.

In one aspect, the sustained release material comprises a polymer suchas polyanhydrides, copolymers of acid and glycolic acid, poly(lactic)acid, poly(glycolic) acid, polyesters, polyorthoesters, proteins,polysaccharides and/or combinations thereof. Preferably, the polymersare biodegradable so that manual removal is avoided. Alternatively, thepolymers are biocompatible and not biodegradable, in those circumstanceswherein it is desirable to physically remove and/or wash out a localanesthetic formulation inserted into a joint space.

The sustained release formulation can contain any quantity of localanesthetic compatible with the selected polymer formulation. Preferably,the local anesthetic is incorporated into the sustained release materialat a percent loading of 0.1% to 90% by weight. Any local anestheticknown to the art may be employed. Preferred local anesthetics includebupivacaine, ropivacaine, dibucaine, etidocaine, tetracaine, lidocaine,xylocaine, mixtures thereof, and/or salts and derivatives thereof.

Augmenting agents useful in potentiating pain relief and/or extendingthe duration of activity include, for example glucocorticosteroids,alphaxalone, allotetrahydrocortisone, aminopyrine, benzamil, clonidine,minoxidil, dehydroepiandrosterone, dextran, diazepam, diazoxide,ouabain, digoxin, spantide, taxol, tetraethylammonium, valproic acid,vincristine, a catecholamine in sustained release form,1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexl]-1-H-pyrrole-2,5-dione,and active derivatives, analogs and mixtures thereof.

Useful glucocorticoid agents include, for example, dexamethasone,cortisone, prednisone, hydrocortisone, beclomethasone dipropionate,betamethasone, flunisolide, methylprednisone, paramethasone,prednisolone, triamcinolone, alclometasone, amcinonide, clobetasol,fludrocortisone, diflorasone diacetate, fluocinolone acetonide,fluocinonide, fluorometholone, flurandrenolide, halcinonide, medrysone.

One skilled in the art will appreciate that an augmenting agent can beoptionally included in the extended duration local anestheticformulation in an amount compatible with, e.g., the extended releasematerial and/or in an amount selected to enhance or prolong the durationof pain relief to the extent desired. For example, an augmenting agent,or combinations of augmenting agents, is incorporated into theformulation substrate at a percent loading ranging from about 0.001% toabout 30% by weight, preferably from about 0.005% to about 15%, byweight.

The augmenting agent is preferably effective to prolong the duration oflocal anesthesia in a treated joint from about 15% to about 1400% of theduration of local anesthesia induced by sustained release localanesthetic without the augmenting agent.

Dextran augmenting agents may have any suitable molecular weight, butpreferably have a molecular weight ranging from about 20 kDa to about200 kDa and are optionally incorporated into said substrate at a percentloading ranging from about 0.01% to about 30% by weight.

In addition, an augmenting agent can include or comprise avasoconstrictor agent in sustained release form. Preferredvasoconstrictor agents include, for example, clonidine, guanfacine,guanabenz, dopa, methyldopa, ephedrine, amphetamine, methamphetamine,methylphenidate, ethylnorepinephrine, ritalin, pemoline, epinephrine,norepinephrine, dopamine, metaraminol, phenylephrine, methoxamine,mephentermine, ephedrine, methysergide, ergotamine, ergotoxine,dihydroergotamine, sumatriptan and analogs, including activemetabolites, derivatives and mixtures of any of the foregoing.

The invention also provides formulations effective to provide localizedpain relief when administered into an intra articular space. Theformulation includes, for example, a local anesthetic incorporated in asustained release formulation, an effective amount of a biocompatiblematerial, and an amount of an augmenting agent effective to prolong theduration of the local anesthesia.

Microparticles according to the invention that are suitable for depositat a site in a patient in need of local anesthesia can optionally beprepared in lyophilized form, e.g., for rehydration prior to use.

The formulation, e.g., in the form of lyophilized particles is alsodesirably prepared in unit dosage form that is sterilized and providedin a container including an amount of such lyophilized particlessufficient to induce prolonged local anesthesia in at least one patientupon suspension in a solution acceptable for deposit into a patient.

Examples demonstrate prolongation of the duration of local anesthesiawith the greater prolongation being provided by the combination of alocal anesthetic with either a glucorticoid or a non-glucocorticoidaugmenting agent.

Preferably, the formulation is in a form suitable for suspension inisotonic saline, physiological buffer or other solution acceptable forinjection into a patient.

In certain preferred embodiments of the invention, the local anestheticis prepared in matrices of biodegradable controlled release injectablemicrospheres. Optionally, the augmenting agent is incorporated intothese matrices along with the local anesthetic.

In further embodiments, a suspension comprising a plurality ofbiocompatible, biodegradable controlled release microspheres comprisinga local anesthetic agent, together with an augmenting agent isincorporated in the controlled release microspheres, or dissolved orsuspended in the suspension of microspheres. The suspension is, forexample, suitable for administering the microspheres by injection.

In yet additional embodiments of the present invention, the localanesthetic is incorporated into a controlled release matrix having theaugmenting agent coated on the surface thereof.

In yet additional embodiments of the invention, the formulationcomprises a local anesthetic core; an augmenting agent present in thecore in an amount effective to prolong the effect of the localanesthetic in an environment of use, and a biocompatible, biodegradablecoating on the core providing a slow release of the local anestheticand/or augmenting agent in an environment of use.

In further embodiments, a portion or all of the local anesthetic isincorporated onto an outer surface of the coated substrate and a portionor all of the augmenting agent is optionally incorporated in the core,so that, e.g., augmenting agent continues to be released after the localanesthetic has dispersed from the controlled release material.

The augmenting agent may be systemically administered by injection orinfiltration, instillation, oral dosing or other method to obtain thedesired prolongation of effect. Systemic administration, (e.g., oral orintravenous) while effective, will require a higher total dose of anaugmentation agent than with local administration in proximity to thelocal anesthetic.

The controlled release local anesthetic dosage form may be injected orinfiltrated, with or without an augmenting agent, at the site where theanesthetic is to be released. This can be prior to surgery, at the timeof surgery, or following removal (discontinuation) or reversal of asystemic anesthetic.

In one preferred embodiment, the formulation is prepared in the form ofmicrospheres. The microspheres may be prepared as a homogenous matrix ofa local anesthetic with a biodegradable controlled release material,with the augmenting agent optionally incorporated therein. Themicrospheres are preferably prepared in sizes suitable for infiltrationand/or injection, and injected at the site where the anesthetic is to bereleased before surgery, during the time of surgery, or followingremoval or reversal of systemic anesthetic.

Examples of intra articular joints where the formulations useful in theinvention can be administered include knee, elbow, hip,sternoclavicular, temporomandibular, carpal, tarsal, wrist, ankle, andany other joint subject to arthritic conditions; examples of bursaewhere the formulations useful in the invention can be administeredinclude acromial, bicipitoradial, cubitoradial, deltoid, infrapatellar,ishchiadica, and other bursa known to those skilled in the art to besubject to pain.

The formulations of the invention are also suitable for administrationin all body spaces/cavities, including but not limited to pleura,peritoneum, cranium, mediastinum, pericardium, bursae or bursal,epidural, intrathecal, intraocular, etc.

The invention is further directed to the use of a formulation comprising(a) controlled release microparticles comprising a local anesthetic andan effective amount of a biocompatible, biodegradable sustained releasematerial prolonging the release of the local anesthetic from theformulation, and (b) a non-toxic augmenting agent in an amount effectiveto prolong the effect of the local anesthetic in-vivo, to treatlocalized joint pain or pain arising from a body space. Preferably, atleast a portion of said augmenting agent is incorporated into saidmicroparticles.

The microparticles are preferably suspended in a pharmaceuticallyacceptable vehicle for injection. The formulation may further comprisean active agent selected from the group consisting of an enzyme, ananti-infective agent, an antibody, a diagnostic aid, a radio-opaque dye,a magnetic resonance imaging dye, a radiolabeled agent, and combinationsthereof. Preferably, at least a portion of said further active agent isincorporated into said microparticles. The local anesthetic ispreferably incorporated into the microparticles at a percent loading of0.1% to 90% by weight. In certain preferred embodiments, the localanesthetic is bupivacaine, the augmenting agent is dexamethasone, andthe sustained release material is a poly(lactide co-glycolide). Infurther preferred embodiments, the microparticles comprise localanesthetic in a percent loading between 0.1% and 90%, preferably between65 and 80%, and augmenting agent is a glucocorticosteroid agent presentin a weight percent relative to the local anesthetic from 0.005% to 15%.

The invention further is directed to the use of a formulation comprising(a) controlled release microparticles comprising a local anesthetic andan effective amount of a biocompatible, biodegradable sustained releasepolymer selected from polyanhydrides, copolymers of lactic acid andglycolic acid, poly(lactic) acid, poly(glycolic) acid, polyesters,polyorthoesters, proteins, polysaccharides and combinations thereof,providing an in-vitro release of said local anesthetic of from 10 to 60percent after 24 hours, from 20 to 80 percent release after 48 hours,and from 40 to 100 percent release after 72 hours; and (b) a non-toxicaugmenting agent in an amount effective to prolong the effect of thelocal anesthetic in-vivo, for providing pain relief a body spaceselected from pleura, peritoneum, cranium, mediastinum, pericardium,bursae or bursal, epidural, intrathecal, and intraocular, or from intraarticular joints selected from knee, elbow, hip, sternoclavicular,temporomandibular, carpal, tarsal, wrist, ankle, and any other jointsubject to arthritic conditions, or from bursae selected from acromial,bicipitoradial, cubitoradial, deltoid, infrapatellar, ishchiadica, andother bursa known to those skilled in the art to be subject to pain, andwhich formulation when administered in-vivo for at least about 24 hours,and preferably for 3-5 days. The formulation may in certain embodimentspreferably comprise a second active agent selected from an enzyme, ananti-infective agent, an antibody, a diagnostic aid, a radio-opaque dye,a magnetic resonance imaging dye, a radiolabeled agent, and combinationsthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 charts for n=2 test animal, the local concentrations verses time,in hours, of bupivacaine concentrations at an intramuscular site of aninjection of extended duration local anesthetic (“EDLA”), in the form ofbupivacaine-containing microspheres. The local concentrations aredetermined by microdialiysis conducted at the site of injection andcalibrated by a perfusion of a known bupivacaine concentration. Thedotted curve shows local concentrations following release of bupivacainefrom EDLA and the solid curve shows local concentrations followinginjection of bupivacaine HCl (1 mg) in immediate release form.

FIG. 2 charts the presence of plasma bupivacaine in each of three testanimals after the injection of EDLA in the form ofbupivacaine-containing microspheres.

DETAILED DESCRIPTION

Accordingly, in a surprising and unexpected finding, methods areprovided for the administration of microparticles in a form suitable forinjection and containing one or more active agents suitable for treatingand/or diagnosing a disease or painful condition in one or morearticular joints in a patient in need thereof. Thus, the inventionprovides a safe and effective procedure for the intra articularadministration of such active agents in sustained release form withoutcausing damage, irritation or inflammation to the treated tissue. Priorto the invention, it was believed that the microparticles might causeinjury and thus be intolerable intra articularly. However, asdemonstrated herein, the microparticles were adequately tolerated. Infurther embodiments, the microparticles are administered into a bodyspace or cavity.

Thus, the present invention provides formulations and methods for thesafe and effective treatment of localized joint conditions by theadministration, e.g., by injection, infusion or infiltration of extendedduration local anesthetic microparticles into an intra articular spaceand/or body space in need of such treatment.

In a preferred aspect, the invention provides methods for relievinglocalized joint pain and/or inflammation. In this aspect of theinvention, the formulations according to the invention include aneffective amount of a local anesthetic agent and preferably an amount ofan augmenting agent, e.g., a glucocorticosteroid or nonglucocorticoidagent that may be provided in any form suitable for intra articularplacement, including forms molded for insertion into a joint space,pastes, solutions and the like. The augmentation of efficacy provided bythe use of the augmenting agent cannot be predicted based on in-vitrorelease (dissolution) of the local anesthetic in sustained release form.The inclusion of the augmenting agent within the sustained releaseformulations of the invention does not substantially alter or prolongthe in-vitro dissolution rate of the local anesthetic agent from theformulation; yet, the same formulation when administered in-vivoprovides a rapid onset of local anesthesia and a significant increase inthe time period of local anesthesia at the site of administration. Theaugmenting agents disclosed herein are both glucocorticoid andnon-glucocorticoid agents and can be administered prior to, along with,or after administration, e.g., topical application, infiltration and/orinjection of the local anesthetic agent in sustained release form, ineach case with a substantial prolongation of local anesthesia in-vivo.

The augmenting agent can be compounded in the same sustained releaseformulation as a local anesthetic agent or agents, in a separatesustained release formulation, e.g., different injectable microspheres,or in a non-sustained release, i.e, immediate release formulation. Theaugmenting agent may be administered before, simultaneously with, orafter injection or infiltration, implantation or insertion of thesustained release local anesthetic formulation at the desired site.

In those embodiments of the invention directed to formulations where theaugmenting agent is included in the formulation with the localanesthetic, the augmenting agent may be included in sustained releaseform or in immediate release form. The augmenting agent may beincorporated into any pharmaceutically acceptable carrier and preferablya carrier providing sustained release, including, e.g., a sustainedrelease matrix along with the local anesthetic; incorporated into asustained release coating on a sustained release device or formulation;or incorporated as an immediate release layer coating the localanesthetic formulation. On the other hand, the augmenting agent may beincorporated into a pharmaceutically acceptable aqueous medium suitablefor infiltration or injection, either in sustained release form or inimmediate release form.

The sustained release formulations and methods of the invention may beused in conjunction with any system for application, infiltration,implantation, insertion, or injection known in the art, including butnot limited to microparticles, e.g., microspheres or microcapsules,gels, pastes, and the like.

As used herein, the terms, “sustained release” and “controlled release”indicate a prolongation of the duration of release and/or duration ofaction of an active agent and are well understood in the art and areintended to be interchangeable, unless otherwise indicated.

As used herein, the term, “active agent” includes, without limitation,any substance that it is desired to incorporate into microparticles forsustained or controlled intra articular delivery and/or release. Anactive agent can be either soluble or insoluble in a polymer solvent andmay be in any state, including liquids, solutions, pastes, solids, andthe like. The active agent may be a pharmaceutically active agent, suchas a drug and/or diagnostic substance for human or veterinary use. Anactive agent can also be an enzyme, antibody, antigen or otherbiological protein or peptide for pharmaceutical and/or diagnostic useor combinations thereof. An active agent may also be, simply by way ofexample, any art known agent, e.g., a polypeptide or peptide derivativeeffective to protect or regenerate cartilage and/or connective tissue.

Additional pharmaceutically active agents that can be incorporated intomicroparticles for intra articular administration, include, e.g.,antibiotics such as sulfisoxazole, penicillin G, ampicillin,cephalosporins, amikacin, gentamicin, tetracyclines, chloramphenicol,erythromycin, clindamycin, isoniazid, rifampin, and derivatives, saltsand mixtures thereof; antifungals such as amphotericin B, nystatin,ketoconazole; antivirals such as acyclovir, amantadine; anticanceragents such as cyclophosphamide, methotrexate, etretinate and other artknown anti-infective or antitumor agents or combinations thereof.

Diagnostic agent that can be administered intra articularly according tothe invention include, e.g., dyes, vital dyes, radio-opaque dyes,magnetic resonance imaging dyes, electron spin dyes, radio-isotopelabeled moieties and others readily apparent to the artisan, orcombinations thereof. In a preferred embodiment, the formulation can beprepared, e.g., to include any art-known nontoxic and radio-opaque dye,e.g., an iodine compound and the like, to aid in the visualization ofthe site for improved accuracy of administration and where desirable, tomonitor the location of any controlled release material remaining at thesite at a later time. In another embodiment, at least a portion of suchoptional radio-opaque dye is present in the suspending vehicle to assistin the localization of the site of injection.

Prodrugs are well known in the art and include inactive drug precursorswhich, when exposed to high temperature, metabolizing enzymes,cavitation and/or pressure, in the presence of oxygen or otherwise, orwhen released from the microspheres, will form active drugs in theintercellular or intracellular environment. Suitable prodrugs will beapparent to those skilled in the art.

Examples of antibodies that can be incorporated into microparticles bythis method generally include industrial antibodies as well asantibodies and derivatives of antibodies for use in biotechnologicalprocess as well as antibodies for diagnostic and therapeutic purposes.Such antibodies include, for example, IgA, IgD, IgG, IgE IgM, andcombinations thereof, in the form of monoclonal, polyclonal andrecombinant antibodies, catalytic antibodies and antigen-bindingantibodies. Further, fragments of antibodies can be incorporated,together with or separately from, intact antibodies. For example,antibody fragments include light and/or heavy chains, and combinationsof light chains or heavy chains, as well as the Fab, Fv, Fc, Fd andsmaller fragments, such as active portions of the variable region andnon-naturally occurring combinations of such fragments and/or light andheavy chains or combinations thereof. Recombinant polypeptides withantibody activity can also be incorporated into microparticles by thismethod, as can engineered antibodies or antibodies or antibody fragmentsthat are linked to other molecules, e.g., drugs, prodrugs and/ordiagnostic or analytic label moieties or combinations thereof.

Examples of genetic materials that can be incorporated, include, e.g.,nucleic acids such as RNA and DNA, of either natural or syntheticorigin, including recombinant RNA and DNA and antisense RNA and DNA aswell as chemical derivatives of these nucleic acids, e.g.,phosphonamides. Types of genetic material that may be incorporatedinclude, for example, genes carried on expression vectors such asplasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), anddefective or “helper” viruses, anti-gene nucleic acids, both single anddouble stranded RNA as well as viral vectors for transforming cells, invivo or in vitro or for genetic therapy, e.g., retroviral vectors,adenoviral vectors and the like or combinations thereof.

Examples of enzymes that can be incorporated into microparticles by thismethod include, generally, enzymes for diagnosis and therapeuticpurposes, e.g., ribonuclease, neuramidinase, trypsin, glycogenphosphorylase, amino peptidase, trypsin chymotrypsin, amylase,muramidase, diesterase, glutamic acid dehydrogenase, as well asfibrinolytic enzymes, lysozymes, dextranase and ribozymes orcombinations thereof, to name but a few that will be readily apparent tothe artisan.

As used herein, the terms “local anesthetic agent” or “local anesthetic”means any drug which provides local numbness and/or analgesia. The termalso includes, but is not limited to, any drug which, when locallyadministered, e.g, topically or by infiltration or injection, provideslocalized full or partial inhibition of sensory perception and/or motorfunction. Under either definition, the localized condition so induced isalso referred to herein as “local anesthesia”. Local anesthesia canresult, for example, from contact of an effective amount of a localanesthetic with sensory nerve processes at the site at which the painfulstimulus is present, or can result from inhibition of nerve transmissionat a nerve or nerves proximal to the site at which the painful stimulusis present.

Local anesthetic agents which can be used include, simply by way ofexample, bupivacaine, ropivacaine, dibucaine, procaine, chloroprocaine,prilocaine, mepivacaine, etidocaine, tetracaine, lidocaine, andxylocaine, as well as anesthetically active derivatives, analogs andmixtures thereof. The local anesthetic can be in the form of a salt, forexample, the hydrochloride, bromide, acetate, citrate, carbonate orsulfate.

More preferably, the local anesthetic agent is in the form of a freebase. The free base provides a slower initial release and avoids anearly “dumping” of the local anesthetic at the injection site. Preferredlocal anesthetic agents include, e.g., bupivacaine. Local anestheticagents typically administered systematically may also be used in thosecases where the means of administration results only in a local effect,rather than systemic.

The term “local anesthetic” may also encompass, pursuant to thedefinitions provided herein, a drug of a different class than thosetraditionally associated with local anesthetic properties, including butnot limited to morphine, fentanyl, and agents which, for example, canprovide regional blockage or localized anesthesia of nociceptivepathways (afferent and/or efferent).

As used herein, the term “microparticles” includes microspheres andmicrocapsules in a size range suitable for injection into a desired siteof administration by injection, infiltration, infusion and the like. Foradministration by injection and/or infiltration or infusion, theformulations according to the invention may be suspended (e.g., formicroparticles), or dissolved (e.g., for immediate release forms), inany art-known vehicle suitable for injection and/or infiltration orinfusion. Such vehicles include, simply by way of example, isotonicsaline, buffered or unbuffered and the like and may optionally includeany other art known ingredients or agents, e.g., colorants,preservatives, antibiotics, epinephrine and other art known ingredients.A more complete listing of art-known vehicles for administration offormulations by systemic administration and/or local injection and/orinfiltration is provided by reference texts that are standard in theart, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 16th Edition,1980 and 17th Edition, 1985, both published by Mack Publishing Company,Easton, Pa., the disclosures of which are incorporated by referenceherein in their entireties.

Formulations according to the invention provide extended duration localanesthetic and may be referred to hereinbelow as “EDLA” formulations.

As used herein, the term “patient” broadly refers to any animal that isto be treated with the compositions and by the methods herein disclosed.The disclosed extended duration micropartical formulations and methodsfor intra articular administration can provide prolonged and effectiveadministration of active agents. In particular, the method for intraarticular administration of extended duration local anesthetic dosageforms according to the invention can provide localized pain blockade toany animal, e.g., any vertebrate, which it is desired to so anesthetize.In particular, the disclosed methods and compositions will find use inveterinary practice and animal husbandry for, e.g., birds and mammals,wherever prolonged local anesthesia is convenient or desirable. In apreferred embodiment, the term “patient” includes humans in need of ordesiring prolonged intra articular treatments, such as for treatment ofjoint pain.

Augmenting Agents

Augmenting agents according to the invention are compositions orcompounds that prolong the duration of local anesthesia and/or enhancethe effectiveness of local anesthetic agents when delivered to the siteof local anesthetic administration before, simultaneously with or afterthe local anesthetic is administered.

In certain embodiments of the invention, the augmenting agent can befrom one or more of the following general types or classes of agents,including glucocorticosteroid agents, alkalinizing agents,non-glucocorticoid steroids such as, e.g, neuroactive steroids and/orsteroid or nonsteroid modulators of gamma amino butyric acid (“GABA”)receptors, modulators of ionic transport across cell membranes,including, e.g., modulators of membrane transport of monovalent anddivalent metal ions such as, for example, blockers or enhancers ofsodium, potassium and/or calcium transport across cell membranes,antipyretic agents, adrenergic receptor agonists or antagonists, such asα2 receptor agonists, tubulin binding agents, including, e.g., agentsthat are capable of either causing formation or disruption ofintracellular microtubules, osmotic polysaccharides, agonists andantagonists of potassium ATP channels, i.e., able to open or closepotassium ATP channels, Na, K-ATPase inhibitors and enhancers,neurokinin antagonists, PLC (i.e., phosphatidylinositol-specificphospholipase C) inhibitors, inhibitors of leukocyte glucose metabolismand anti-convulsants. The augmenting agent can also be an analeptic, atranquilizing agent, an ataretic, an antidepressant, an anti-seizureagent, leukotriene and prostaglandin agonists and inhibitors,phosphodiesterase agonists and inhibitors, e.g., based on cAMP, andcombinations of any of the foregoing. Vasoconstrictive agents providedin controlled release form also provide for unexpected and surprisingaugmentation of duration and potency of local anesthetics relative toimmediate release forms of vasonstrictive agents heretofore known to theart. The aforementioned types of augmenting agents may to used alone orin any mixture or combination of each such agent to provide effectiveaugmentation of local anesthesia where desired.

In one embodiment, the augmenting agent is any art-knownglucocorticosteroid agent, such as, simply by way of example,dexamethasone, cortisone, prednisone, hydrocortisone, beclomethasonedipropionate, betamethasone, flunisolide, methylprednisone,paramethasone, prednisolone, triamcinolone, alclometasone, amcinonide,clobetasol, fludrocortisone, diflorasone diacetate, fluocinoloneacetonide, fluocinonide, fluorometholone, flurandrenolide, halcinonide,medrysone and mometasone, ropivicaine and pharmaceutically acceptablemixtures and salts thereof and any other derivatives and analogsthereof.

When a glucocorticosteroid agent is included in the controlled releasesubstrates comprising local anesthetic, it has been found that usefulloadings of glucocorticosteroid agent are, e.g., from 0.005% to 30% byweight of the substrate.

When the glucocorticosteroid agent is included with a suitable vehiclein which microparticles comprising local anesthetic are suspended, theglucocorticosteroid agent is present, for example, in a weight percentrelative to the local anesthetic varying from about 0.005% to about 15%.

In another embodiment, the augmenting agents include an alkalinizingagent. The alkalinizing augmenting agents used herein preferably raisethe pH of the medium in which the local anesthetic agents in sustainedrelease form are present (e.g., either an injection medium or theenvironment at the site of injection) to provide a pH from about 6.0 toabout 8.5, preferably from about 7.5 to about 8.5. Preferably, thealkalinizing agent may be, for example, a carbonate buffer such assodium carbonate. Of course, any other alkalinizing agent that ispharmaceutically acceptable for localized injection or infiltration mayalso be effectively employed. The augmenting agents also includenon-glucocorticoid steroids such as e.g., androgens, such astestosterone and its active derivatives, analogs and metabolites;estrogens, such as estradiol and its active derivatives, analogs andmetabolites and progestins, such as progesterone and its activederivatives, analogs and metabolites and mixtures of any of these.

In yet another embodiment, the augmenting agents are neuroactivesteroids, such as, e.g., one or more of the class of anestheticsteroids. Neuroactive steroids useful as augmenting agents according tothe invention also include those which modulate GABA receptors.Preferred neuroactive steroids include, simply by way of example,althesin and its main component, alphaxalone and active analogs,derivatives and mixtures thereof, as well as 5-alpha-pregnane-3alpha-21-diol-20-one (tetrahydrodeoxycorticosterone or THDOC) and/orallotetrahydrocortisone (the 17-beta configuration); anddehydroepiandrosterone (“DHE”) and active analogs, derivatives andmixtures thereof. Preferably, the neuroactive steroids are present as anadditive in the vehicle carrying the microspheres in a concentrationranging from about 0.01% to about 1% by weight, and most preferably fromabout 0.05% to about 0.5% by weight.

The augmenting agents also include non-steroidal modulators of GABAreceptors, including those that are capable of potentiating theinhibitory effects of GABA on those receptors. Preferably, these includethe benzodiazepines, e.g., diazepam as well as its active derivatives,analogs and metabolites and mixtures thereof. More preferably, thediazepam is present as an additive in the vehicle in a concentrationranging from about 0.01% to about 1% by weight, and most preferably fromabout 0.05% to about 0.5% by weight. Of course, the artisan willappreciate that the potency of benzodiazepines varies widely, and willadjust these concentration ranges accordingly for other benzodiazepines,relative to the potency of diazepam.

In yet another aspect of the invention, the augmenting agent is amodulator of ionic transport across cell membranes. Monovalent andmultivalent metal ion transport can be modulated. Agents include, e.g.,sodium, potassium and calcium channel modulators (e.g., nifedipine,nitrendipine, verapamil, etc.). In preferred embodiments, these alsoinclude, but are not limited to, aminopyridine, benzamil, diazoxide, 5,5diphenylhydantoin, minoxidil, tetrethylammonium and valproic acid.Preferably, the ion transport modulating agent is present as an additivein the vehicle carrying the microspheres in a concentration ranging fromabout 0.01 to about 5 percent by weight, and most preferably from about0.05 to about 1.5 percent by weight.

Augmenting agents also include, e.g., antipyretic agents such asaminopyrine, phenazone, dipyrone, apazone, phenylbutazone andderivatives and analogs thereof. Aminopyrine is preferably included inthe vehicle containing the microspheres in a concentration ranging fromabout 0.01 to about 0.5 percent and in a more preferred embodiment theconcentration ranges from about 0.05 to about 0.5 percent, by weight.

Other preferred augmenting agents include, e.g., adrenergic receptormodulators, such as α2 receptor agonists, can also be used as augmentingagents. Simply by way of example, the α2 receptor agonist clonidineprovides useful augmentation of local anesthesia, although any other artknown α2 receptor modulators capable of augmenting local anesthesiaaccording to the invention may be used. Clonidine is preferably includedin the vehicle containing the microspheres in a concentration rangingfrom about 0.01% to about 0.5% preferred embodiment the concentrationranges from about 0.05% to about 1%, by weight.

Tubulin binding agents that are capable of promoting the formation ordisruption of cytoplasmic microtubules may be employed as augmentingagents according to the invention. Such agents include, for example,colchicine and the vinca alkaloids (vincristine and vinblastine) as wellas active derivatives, analogs metabolites and mixtures thereof. Ofcourse, some agents may be classified in more than one category, thus,for example, colchicine is also known to inhibit glucose metabolism inleukocytes. Colchicine is preferably included in the vehicle containingthe microspheres in a concentration ranging from about 0.01 to about 1.0percent and in a more preferred embodiment the concentration ranges fromabout 0.05 to about 0.5 percent, by weight.

Osmotic polysaccharides are also able to be used as augmenting agents.In one preferred embodiment, the osmotic polysaccharide includesdextran. More preferably, the dextran augmenting agents according to theinvention have a molecular weight ranging from about 20 kDa throughabout 200 kDa, or greater. A solution containing dextran in a formsuitable for injection or infiltration into a desired site in a patientis preferably buffered to a pH ranging from about 3.0 to about 8.5, butin a preferred aspect is buffered to a pH ranging from about 7.0 toabout 8.5.

Other preferred embodiments of the invention provide for potassium-ATPchannel agonists for use as augmenting agents. A preferred potassium-ATPchannel agonist is, e.g., diazoxide, as well as its active derivatives,analogs, metabolites and mixtures thereof which are useful as augmentingagents.

Sodium/potassium ATPase inhibitors are also preferred as augmentingagents according to the invention. Preferably, the sodium/potassiumATPase inhibitors are cardiac glycosides that are effective to augmentlocal anesthesia. Cardiac glycosides that are useful according to theinvention include, e.g., oubaine, digoxin, digitoxin and activederivatives, analogs and metabolites and mixtures of any of these.

Additionally, augmenting agents according to the invention include,e.g., neurokinin antagonists, such as, e.g., spantide and other peptideinhibitors of substance P receptors that are well known to the art,e.g., as are listed in Receptor and Ion Channel Nomenclature Supplement,Trends in Pharmacological Sciences 18:64-65, the disclosure of which isincorporated by reference herein in its entirety. PLC (i.e.,phosphatidylinositol-specific phospholipase C) inhibitors such as, e.g.,1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexl]-1-H-pyrrole-2,5-dione, and anti-seizure agents andagents that stabilize cell membrane potential, such as, e.g.,benzodiazepines, barbiturates, deoxybarbiturates, carbamazepine,succinamides, valproic acid, oxazalidienbiones, phenacemide and activederivatives, analogs and metabolites and mixtures thereof. Preferably,the anti-seizure augmenting agent is phenytoin, and most preferably is5,5-diphenylhydantoin.

Surprisingly, locally acting vasoconstrictive agents also provideeffective augmentation of local anesthesia that is unexpectedly superiorto that provided by immediate release vasoconstrictive agents. While notwishing to be bound by any hypothesis as to how vasconstrictive agentsin sustained release form might greatly prolong local anestheticactivity, it is believed that sustained release vasoconstrictor agentsprovide a controlled and non-toxic vasoconstrictor activity that reducesthe rate of local anesthetic washout from the treated tissue area toprolong the presence of effective concentrations of local anesthetic inthe tissue. It is known to the art that vasoconstrictors, e.g.,epinephrine, prolong local anesthetic activity for, at best, about 1hour and that if excessive amounts of epinephrine or othervasoconstrictor is administered in an attempt to further prolong localanesthesia, local circulation may be so disrupted as to cause tissuenecrosis and gangrene.

It is therefore unexpected that sustained release vasoconstrictor agentscan achieve local tissue concentrations that are safe and effective toprovide vasoconstrictor activity effective to substantially prolonglocal anesthesia. More unexpectedly, the local circulatory bed, i.e.,blood vessels, remain responsive to the vasoconstrictor agent forprolonged periods, e.g., receptor desensitization or smooth musclefatigue or tolerance does not prevent the prolongation effect. Thegradual release from a sustained release formulation also serves togreatly reduce the risk of toxic reactions such as, e.g., localizedtissue necroses.

The previously discussed vasoconstrictive augmenting agents can beadministered before, simultaneously with or after the administration oflocal anesthetic. In one embodiment of the invention, at least a portionof the vasoconstrictive agent is formulated in a sustained releaseformulation together with local anesthetic. In another embodiment, thevasconstrictive agent is prepared in one or separate sustained releaseformulations. It will be appreciated that by manipulating the loadingof, e.g., microspheres containing vasoconstrictor agent, the artisan candetermine the number of microspheres necessary to administer a givendose. Thus, simply by way of example, microspheres loaded with about 75percent by weight of vasoconstrictor agent will require half of themicrospheres necessary to administer a predetermined dose than willmicrospheres loaded with about 45 percent by weight of vasoconstrictoragent.

Vasoconstrictor agents can formulated into, e.g., sustained releasemicrospheres including both a local anesthetic, e.g., bupivacaine freebase, and a vasoconstrictor agent. Vasoconstrictor agents can also beformulated into, e.g., sustained release microspheres including localanesthetic without a vasoconstrictive agent.

While not wishing to be bound by any hypothesis as to howvasconstrictive agents in sustained release form might greatly prolonglocal anesthetic activity, it is believed that sustained releasevasoconstrictor agents provide a controlled and non-toxicvasoconstrictor activity that reduces the rate of local anestheticwashout from the treated tissue area to prolong the presence ofeffective concentrations of local anesthetic in the tissue. It is knownto the art that vasoconstrictors, e.g., epinephrine, prolong localanesthetic activity for, at best, about 1 hour and that if excessiveamounts of epinephrine or other vasoconstrictor is administered in anattempt to further prolong local anesthesia, local circulation may be sodisrupted as to cause tissue necrosis and gangrene.

It is therefore unexpected that sustained release vasoconstrictor agentscan achieve local tissue concentrations that are safe and effective toprovide vasoconstrictor activity effective to substantially prolonglocal anesthesia. More unexpectedly, the local circulatory bed, i.e.,blood vessels, remain responsive to the vasoconstrictor agent forprolonged periods, e.g., receptor desensitization or smooth musclefatigue or tolerance does not prevent the prolongation effect. Thegradual release from a sustained release formulation also serves togreatly reduce the risk of toxic reactions such as, e.g., localizedtissue necroses.

Vasoconstrictor agents can formulated into, e.g., sustained releasemicrospheres including both a local anesthetic, e.g., bupivacaine freebase, and a vasoconstrictor agent. Vasoconstrictor agents can also beformulated into, e.g., sustained release microspheres including localanesthetic without a vasoconstrictive agent.

In one embodiment, local anesthetic and vasoconstrictor agents areadministered simultaneously in the form of, e.g., separate microspheressuspended in a single medium suitable for injection or infiltration, orin separate microspheres suitable for injection, e.g., at the same site.In a further embodiment, simply by way of example, administration ofsustained release microspheres with combined local anesthetic andvasoconstrictor agent can also be followed by one or more additionaladministrations of such combination formulation and/or of microspheresincluding as the active agent only local anesthetic or onlyvasoconstrictor agent.

Augmenting agents that are vasoconstrictor agents in sustained releaseform include, but are not limited to, catecholamines e.g., epinephrine,norepinephrine and dopamine as well as, e.g., metaraminol,phenylephrine, methoxamine, mephentermine, methysergide, ergotamine,ergotoxine, dihydroergotamine, sumatriptan and analogs, and alpha-1 andalpha-2 adrenergic agonists, such as, e.g., clonidine, guanfacine,guanabenz and dopa (i.e., dihyrdoxyphenylalanine), methyldopa,ephedrine, amphetamine, methamplietamine, methylphenidate,ethylnorepinephrine ritalin, pemoline and other sympathomimetic agents,including active metabolites, derivatives and mixtures of any of theforegoing.

In a more preferred embodiment, at least a portion of any of theaugmenting agents enumerated above are included in the sustained releaseformulation, in combination with a local anesthetic agent or agents in aconcentration ranging from about 0.01 to about 30 percent or more, byweight, relative to the weight of the formulation.

The artisan will also appreciate that other augmenting agents accordingto the invention broadly include any other types and classifications ofdrugs or active agents known to the art. Such augmenting agents arereadily identified by routine screening as discussed hereinbelow usinganimal sensory and motor quantitation protocols well known to the art.

A local anesthetic according to the invention can also be formulated,e.g., in injectable microspheres, in combination with at least onevasoconstrictor augmenting agent according to the invention. In oneembodiment, the vasoconstrictor can be included in the vehicle suitablefor injection carrying the microspheres. In a further embodiment, atleast a portion of the vasoconstrictor can also be formulated into asustained release formulation, e.g., injectable microspheres, togetherwith the local anesthetic. In a still further embodiment, at least aportion of the vasoconstrictor can be prepared in a separate sustainedrelease formulation.

The vasoconstrictor can be included in either a single or combinationformulation in an amount ranging from about 0.001 percent to about 90percent, by weight relative to the total weight of the formulation.Preferably, the vasoconstrictor is included in a sustained releaseformulation in an amount ranging from about 0.005 percent to about 20%,and more preferably, from about 0.05 percent to about 5 percent, byweight, relative to the total weight of the formulation. When avasoconstrictor is present in the injection vehicle in immediate releaseform, it is present in amounts ranging from about 0.01% to about 5percent, or more, by weight, relative to the injection vehicle. Thevasoconstrictor can also be provided in a ratio of local anesthetic,e.g., bupivacaine to vasoconstrictor, ranging from about 10:1 to about20,000 and preferably from about 100:1 to about 2000:1 and from about500:1 to about 1500:1.

Of course, the artisan will appreciate that the amounts of augmentingagent and local anesthetic will vary depending upon the relative potencyof the agents selected, the depth and duration of local anesthesiadesired.

Of course, the artisan will appreciate that the optimal concentrationand/or quantities or amounts of any particular augmenting agent, whetherpresent in the injection vehicle, separately administered before, duringor after local anesthesia is induced or whether included in themicrosphere formulation, may be adjusted to accommodate variations inthe treatment parameters. Such treatment parameters include the polymercomposition of a particular microsphere preparation, the particularlocal anesthetic utilized, and the clinical use to which the preparationis put, in terms of the site treated for local anesthesia, the type ofpatient, e.g., human or non-human, adult or child, and the type ofsensory stimulus to be anesthetized.

Further, the concentration and/or amount of any particular augmentingagent for a given formulation may be readily identified by routinescreening in animals, e.g, rats, by screening a range of concentrationand/or amounts of augmenting agent using the hotplate foot withdrawalassay and/or motor function assay described hereinbelow.

Art known methods are also available to assay local tissueconcentrations, diffusion rates from microspheres and local blood flowbefore and after administration of local anesthetic formulationsaccording to the invention. One such method is microdialysis, asreviewed by T. E. Robinson et al., 1991, MICRODIALYSIS IN THENEUROSCIENCES, Techniques, volume 7, Chapter 1, pages 1-64, incorporatedherein by reference in its entirety.

The methods reviewed by Robinson can be applied, in brief, as follows. Amicrodialysis loop is placed in situ in a test animal. Dialysis fluid ispumped through the loop. When microspheres according to the inventionare injected adjacent to the loop, released drugs, e.g., bupivacaine andvasoconstrictor augmenting agents, are collected in the dialysate inproportion to their local tissue concentrations. The progress ofdiffusion of the active agents can be determined thereby with suitablecalibration procedures using known concentrations of active agents. Forthe vasoconstrictor augmenting agents, decrements and durations ofvasoconstriction effects can be measured by clearance rates of markersubstances, e.g., methylene blue or radiolabeled albumen from the localtissue.

The data presented hereinbelow by the Examples applies microdialysis toconfirm that bupivacaine containing microspheres placed into tissueprovides an initial rise of free bupivacaine, followed by the prolongedmaintenance of high local concentration.

The optimal concentration of augmenting agent for human clinical use mayalso be readily determined by routine animal screening as describedhereinbelow, and further adjusted, where indicated, by routine clinicalexperience.

Formulations

Any pharmaceutically acceptable vehicle or formulation suitable forlocal infiltration or injection into a site to be anesthetized, that isable to provide a sustained release of an active agent may be employedto provide for prolonged local anesthesia as needed. Slow releaseformulations known in the art include specially coated pellets, polymerformulations or matrices for surgical insertion or as sustained releasemicroparticles, e.g., microspheres or microcapsules, for implantation,insertion or injection, wherein the slow release of the activemedicament is brought about through sustained or controlled diffusionout of the matrix and/or selective breakdown of the coating of thepreparation or selective breakdown of a polymer matrix. Otherformulations or vehicles for sustained or immediate delivery of an agentto a preferred localized site in a patient include, e.g., suspensions,emulsions, liposomes and any other suitable, art known, delivery vehicleor formulation.

In a preferred embodiment, the slow release formulation is prepared asmicrospheres in a size distribution range suitable for localinfiltration or injection. The diameter and shape of the microspheres orother particles can be manipulated to modify the releasecharacteristics. For example, larger diameter microspheres willtypically provide slower rates of release and reduced tissue penetrationand smaller diameters of microspheres will produce the opposite effects,relative to microspheres of different mean diameter but of the samecomposition. In addition, other particle shapes, such as, for example,cylindrical shapes, can also modify release rates by virtue of theincreased ratio of surface area to mass inherent to such alternativegeometrical shapes, relative to a spherical shape. The diameter ofinjectable microspheres are in a size range, for example, from about 5microns to about 200 microns in diameter. In a more preferredembodiment, the microspheres range in diameter from about 20 to about120 microns.

A wide variety of biocompatible materials may be utilized to provide thecontrolled/sustained release of the local anesthetic. Anypharmaceutically acceptable biocompatible polymers known to thoseskilled in the art may be utilized. It is preferred that thebiocompatible sustained release material degrade in-vivo over a periodof less than about two years, with at least 50% of the sustained releasematerial degrading within about one year, and more preferably six monthsor less. More preferably, the sustained release material will degradesignificantly within one to three months, with at least 50% of thematerial degrading into non-toxic residues which are removed by thebody, and 100% of the drug being released within a time period fromabout two weeks to about two months. A degradable sustained releasematerial should preferably degrade by hydrolysis, and most preferably bysurface erosion, rather than by bulk erosion, so that release is notonly sustained but also provides desirable release rates. However, thepharmacokinetic release profile of these formulations may be firstorder, zero order, bi- or multi-phasic, to provide the desiredreversible local anesthetic effect over the desired time period.

In the case of polymeric materials, biocompatibility is enhanced byrecrystallization of either the monomers forming the polymer and/or thepolymer using standard techniques.

Suitable biocompatible polymers can be utilized as the sustained releasematerial. The polymeric material may comprise biocompatible,biodegradable polymers such as a polylactide, a polyglycolide, apoly(lactide-co-glycolide), a polyanhydride, a polyorthoester,polycaprolactones, polyphosphazenes, polysaccharides, proteinaceouspolymers, soluble derivatives of polysaccharides, soluble derivatives ofproteinaceous polymers, polypeptides, polyesters, and polyorthoesters ormixtures or blends of any of these. The polysaccharides may bepoly-1,4-glucans, e.g., starch glycogen, amylose, amylopectin, andmixtures thereof. The biodegradable hydrophilic or hydrophobic polymermay be a water-soluble derivative of a poly-1,4-glucan, includinghydrolyzed amylopectin, hydroxyalkyl derivatives of hydrolyzedamylopectin such as hydroxyethyl starch (HES), hydroxyethyl amylose,dialdehyde starch, and the like. Preferred sustained release materialswhich are useful in the formulations of the invention include thepolyanhydrides, co-polymers of lactic acid and glycolic acid wherein theweight ratio of lactic acid to glycolic acid is no more than 4:1 (i.e.,80% or less lactic acid to 20% or more glycolic acid by weight), andpolyorthoesters containing a catalyst or degradation enhancing compound,for example, containing at least 1% by weight anhydride catalyst such asmaleic anhydride. Other useful polymers include protein polymers such asgelatin and fibrin and polysaccharides such as hyaluronic acid. Sincepolylactic acid takes at least one year to degrade in-vivo, this polymershould be utilized by itself only in circumstances where such adegradation rate is desirable or acceptable.

The polymeric material may be prepared by any method known to thoseskilled in the art. For example, where the polymeric material iscomprised of a copolymer of lactic and glycolic acid, this copolymer maybe prepared by the procedure set forth in U.S. Pat. No. 4,293,539(Ludwig, et al.), the disclosure of which is hereby incorporated byreference in its entirety. In brief, Ludwig prepares such copolymers bycondensation of lactic acid and glycolic acid in the presence of areadily removable polymerization catalyst (e.g., a strong acidion-exchange resin such as Dowex HCR-W2-H). The amount of catalyst isnot critical to the polymerization, but typically is from about 0.01 toabout 20 parts by weight relative to the total weight of combined lacticacid and glycolic acid. The polymerization reaction may be conductedwithout solvents at a temperature from about 100° C. to about 250° C.for about 48 to about 96 hours, preferably under a reduced pressure tofacilitate removal of water and by-products. The copolymer is thenrecovered by filtering the molten reaction mixture to removesubstantially all of the catalyst, or by cooling and then dissolving thereaction mixture in an organic solvent such as dichloromethane oracetone and then filtering to remove the catalyst.

Various commercially available poly (lactide-co-glycolide) materials(PLGA) may be used in the preparation of the microspheres of the presentinvention. For example, poly(d,l-lactic-co-glycolic acid) arecommercially available from Medisorb Technologies International L.P.(Cincinnati, Ohio). A preferred product commercially available fromMedisorb is a 50:50 poly (D,L) lactic co-glycolic acid known as MEDISORB5050 DL. This product has a mole percent composition of 50% lactide and50% glycolide. Other suitable commercially available products areMedisorb 65:35 DL, 75:25 DL, 85:15 DL and poly(d,l-lactic acid)(d,l-PLA). Poly(lactide-co-glycolides) are also commercially availablefrom Boerhinger Ingelheim (Germany) under its Resomer© mark, e.g., PLGA50:50 (Resomer RG 502), PLGA 75:25 (Resomer RG 752) and d,l-PLA (resomerRG 206), and from Birmingham Polymers (Birmingham, Ala.). Thesecopolymers are available in a wide range of molecular weights and ratiosof lactic to glycolic acid.

Pharmaceutically acceptable polyanhydrides which are useful in thepresent invention have a water-labile anhydride linkage. The rate ofdrug release can be controlled by the particular polyanhydride polymerutilized and its molecular weight. The polyanhydride polymer may bebranched or linear. Examples of polymers which are useful in the presentinvention include homopolymers and copolymers of poly(lactic acid)and/or poly(glycolic acid), poly[bis(p-carboxyphenoxy)propane anhydride](PCPP), poly[bis(p-carboxy)methane anhydride] (PCPM), polyanhydrides ofoligomerized unsaturated aliphatic acids, polyanhydride polymersprepared from amino acids which are modified to include an additionalcarboxylic acid, aromatic polyanhydride compositions, and co-polymers ofpolyanhydrides with other substances, such as fatty acid terminatedpolyanhydrides, e.g., polyanhydrides polymerized from monomers of dimersand/or trimers of unsaturated fatty acids or unsaturated aliphaticacids. Polyanhydrides may be prepared in accordance with the methods setforth in U.S. Pat. No. 4,757,128, hereby incorporated by reference. Forexample, polyanhydrides may be synthesized by melt polycondensation ofhighly pure dicarboxylic acid monomers converted to the mixed anhydrideby reflux in acetic anhydride, isolation and purification of theisolated prepolymers by recrystallization, and melt polymerization underlow pressure (10⁻⁴ mm) with a dry ice/acetone trap at a temperaturebetween 140° C.-250° C. for 10-300 minutes. High molecular weightpolyanhydrides are obtained by inclusion of a catalyst which increasesthe rate of anhydride interchain exchange, for example, alkaline earthmetal oxides such as CaO, BaO and CaCO₃. Polyorthoester polymers may beprepared, e.g., as set forth in U.S. Pat. No. 4,070,347, herebyincorporated by reference.

Proteinaceous polymers may also be used. Proteinaceous polymers andtheir soluble derivatives include gelation biodegradable syntheticpolypeptides, elastin, alkylated collagen, alkylated elastin, and thelike. Biodegradable synthetic polypeptides includepoly-(N-hydroxyalkyl)-L-asparagine, poly-(N-hydroxyalkyl)-L-glutamine,copolymers of N-hydroxyalkyl-L-asparagine and N-hydroxyalkyl-L-glutaminewith other amino acids. Suggested amino acids include L-alanine,L-lysine, L-phenylalanine, L-valine, L-tyrosine, and the like.

In embodiments where the biodegradable polymer comprises a gel, one suchuseful polymer is a thermally gelling polymer, e.g., polyethylene oxide,polypropylene oxide (PEO-PPO) block copolymer such as Pluronic® F127from BASF Wyandotte. In such cases, the local anesthetic formulation maybe injected via syringe as a free-flowing liquid, which gels rapidlyabove 30° C. (e.g., when injected into a patient). The gel system thenreleases a steady dose of local anesthetic at the site ofadministration.

In additional embodiments, the sustained release material, which ineffect acts as a carrier for the local anesthetic and/or the augmentingagent, can further include a bioadhesive polymer such as pectins(polygalacturonic acid), mucopolysaccharides (hyaluronic acid, mucin) ornon-toxic lectins or the polymer itself may be bioadhesive, e.g.,polyanhydride or polysaccharides such as chitosan.

Definitions or further descriptions of any of the foregoing terminologyare well known in the art and may be found by referring to any standardbiochemistry reference text such as “Biochemistry” by Albert L.Lehninger, Worth Publishers, Inc. and “Biochemistry” by Lubert Stryer,W.H. Freeman and Company, both of which are hereby incorporated byreference.

The aforementioned biodegradable hydrophobic and hydrophilic polymersare particularly suited for the methods and compositions of the presentinvention by reason of their characteristically low human toxicity andvirtually complete biodegradability.

The substrates of the presently described formulations in certainpreferred embodiments are manufactured using a method that evenlydisperses the local anesthetic throughout the formulation, such asemulsion preparation, solvent casting, spray drying or hot melt, ratherthan a method such as compression molding. A desired release profile canbe achieved by using a mixture of polymers having different releaserates and/or different percent loading of local anesthetic and/oraugmenting agent, for example, polymers releasing in one day, threedays, and one week. In addition, a mixture of microspheres having one ormore different local anesthetic agents, having the same or differentcontrolled release profile, can be utilized to provide the benefits ofdifferent potencies and spectrum of activity during the course oftreatment.

Methods for manufacture of microspheres are well known and are typifiedin the following examples. Examples of suitable methods of makingmicrospheres include solvent evaporation, phase separation and fluidizedbed coating.

In solvent evaporation procedures, the local anesthetic agent, ifsoluble in organic solvents, may be entrapped in the biodegradablepolymer by dissolving the polymer in a volatile organic solvent, addingthe drug to the organic phase, emulsifying the organic phase in waterwhich contains less than 2% polyvinyl alcohol, and finally removing thesolvent under vacuum to form discrete, hardened monolithic microspheres.

Phase separation microencapsulation procedures are suitable forentrapping water-soluble agents in the polymer to prepare microcapsulesand microspheres. Phase separation involves coacervation of the polymerfrom an organic solvent by addition of a nonsolvent such as siliconeoil. In a preferred embodiment, the microspheres may be prepared by theprocess of Ramstack et al., 1995, in published international patentapplication WO 95/13799, the disclosure of which is incorporated hereinin its entirety. The Ramstack et al. process essentially provides for afirst phase, including an active agent and a polymer, and a secondphase, that are pumped through a static mixer into a quench liquid toform microparticles containing the active agent. The first and secondphases can optionally be substantially immiscible and the second phaseis preferably free from solvents for the polymer and the active agentand includes an aqueous solution of an emulsifier. In fluidized bedcoating, the drug is dissolved in an organic solvent along with thepolymer. The solution is then processed, e.g., through a Wurster airsuspension coating apparatus to form the final microcapsule product.

The biodegradable sustained release materials may be used in order toprepare sustained release local anesthetic implants. The implants may bemanufactured, e.g., by compression molding, injection molding, and screwextrusion, whereby the local anesthetic agent is loaded into thepolymer. Implantable fibers can be manufactured, e.g., by blending thelocal anesthetic agent with the sustained release material and thenextruding the mixture, e.g., under pressure, to thereby obtainbiodegradable fibers. In certain preferred embodiments, the augmentingagent may be incorporated into the implant, or may be coated onto asurface of the implant.

In other embodiments of the invention, the sustained release materialcomprises an artificial lipid vesicle, or liposome. The use of liposomesas drug delivery systems is known, and comprehensive review articles ontheir properties and clinical applications are available; see, e.g.,Barenholz and Amselem, in “Liposome Technology”, 2nd ed., G.Gregoriadis, ed., CRC Press, 1992; Lichtenberg and Barenholz, in Methodsfor Biochemical Analysis, 33, D. Glick, ed., 1988. A liposome is definedas a structure consisting of one or more concentric lipid bilayersseparated by water or aqueous buffer compartments. These hollowstructures, which have an internal aqueous compartment, can be preparedwith diameters ranging from 20 nm to 10 μm. They are classifiedaccording to their final size and preparation method as: SUV, smallunilamellar vesicles (0.5-50 nm); LUV, large unilamellar vesicles (100nm); REV, reverse phase evaporation vesicles (0.5 μm); and MLV, largemultilamellar vesicles (2-10 μm).

Liposomes as described herein will vary in size. Preferably, theliposomes have a diameter between 100 nm and 10 microns or greater. Awide variety of lipid materials may be used to form the liposomesincluding natural lecithins, e.g., those derived from egg and soya bean,and synthetic lecithins, the proviso being that it is preferred that thelipids are non-immunogenic and biodegradable. Also, lipid-basedmaterials formed in combination with polymers may be used, such as thosedescribed in U.S. Pat. No. 5,188,837 to Domb, (incorporated by referenceherein).

Examples of synthetic lecithins which may be used together with theirrespective phase transition temperatures, aredi-(tetradecanoy)phosphatidylcholine (DTPC) (23 C),di-(hexadecanoyl)phosphatidylcholine (DHPC) (41 C) anddi-(octandecanoyl) phosphatidylcholine (DOPC) (55 C). Di-(hexadecanoyl)phosphatidylcholine is preferred as the sole or major lecithin,optionally together with a minor proportion of the di-(octadecanoyl) orthe di-(tetradecanoyl) compound. Other synthetic lecithins which may beused are unsaturated synthetic lecithins, for example,di-(oleyl)phosphatidyl-choline and di-(linoleyl)phosphatidylcholine. Inaddition to the main liposome-forming lipid or lipids, which are usuallyphospholipids, other lipids (e.g. in a proportion of 5-40% w/w of thetotal lipids) may be included, for example, cholesterol or cholesterolstearate, to modify the structure of the liposome membrane, rendering itmore fluid or more rigid depending on the nature of the mainliposome-forming lipid or lipids.

In certain embodiments, the augmenting agent is incorporated along withthe local anesthetic agent into the lipid. In other preferredformulations, the lipids containing the local anesthetic agent aredispersed in a pharmaceutically acceptable aqueous medium. Theaugmenting agent may be incorporated into this aqueous medium. In afurther embodiment, a portion of the dose of the local anesthetic isincorporated into the aqueous medium in immediate release form. Theresultant formulation is an aqueous suspension which may comprise thelocal anesthetic and/or augmenting agent partitioned between a freeaqueous phase and a liposome phase.

As an even further alternate embodiment, liposomes containing localanesthetic may be combined in an aqueous phase where liposomescontaining the augmenting agent form an aqueous pharmaceuticalsuspension useful for administration at the desired site in the patientto be anesthetized. This may be accomplished via injection orimplantation. Liposomes may be prepared by dissolving an appropriateamount of a phospholipid or mixture or phospholipids together with anyother desired lipid soluble components (e.g., cholesterol, cholesterolstearate) flowing in a suitable solvent (e.g., ethanol) and evaporatingto dryness. An aqueous solution of the local anesthetic, optionally withaugmenting agent, may then be added and mixed until a lipid film isdispersed. The resulting suspension will contain liposomes ranging insize, which may then fractionated to remove undesirable sizes, ifnecessary. This fractionation may be effected by column gelchromatography, centrifugation, ultracentrifugation or by dialysis, aswell known in the art.

The above method of preparation of liposomes is representative of apossible procedure only. Those skilled in the art will appreciate thatthere are many different methods of preparing liposomes, all of whichare deemed to be encompassed by the present disclosure.

In additional embodiments of the invention, the substrate comprises aplurality of microcapsules laden with the local anesthetic agent with orwithout the augmenting agent. Microcapsules may be prepared, forexample, by dissolving or dispersing the local anesthetic agent in anorganic solvent and dissolving a wall forming material (polystyrene,alkylcelluloses, polyesters, polysaccharides, polycarbonates,poly(meth)acrylic acid ester, cellulose acetate,hydroxypropylmethylcellulose phthalate, dibutylaminohydroxypropyl ether,polyvinyl butyral, polyvinyl formal, polyvinylacetaldiethylaminoacetate, 2-methyl-5-vinyl pyridine methacrylate-methacrylic acidcopolymer, polypropylene, vinylchloride-vinylacetate copolymer, glyceroldistearate, etc.) in the solvent; then dispersing the solvent containingthe local anesthetic agent and wall forming material in acontinuous-phase processing medium, and then evaporating a portion ofthe solvent to obtain microcapsules containing the local anestheticagent in suspension, and finally, extracting the remainder of thesolvent from the microcapsules. This procedure is described in moredetail in U.S. Pat. Nos. 4,389,330 and 4,530,840, hereby incorporated byreference.

The sustained release dosage forms of the present invention preferablyprovide a sustained action in the localized area to be treated. Forexample, it would be desirable that such a formulation provideslocalized anesthesia to the site for a period of one day, two days,three days, or longer. The formulations can therefore, of course, bemodified in order to obtain such a desired result.

Microspheres and other injectable substrates described herein may beincorporating an effective amount of the same into a pharmaceuticallyacceptable solution (e.g., water) or suspension for injection. The finalreconstituted product viscosity may be in a range suitable for the routeof administration. In certain instances, the final reconstituted productviscosity may be, e.g., about 35 cps. Administration may be via thesubcutaneous or intramuscular route. However, alternative routes arealso contemplated, and the formulations may be applied to the localizedsite in any manner known to those skilled in the art, such that alocalized effect is obtained. The substrate formulations of theinvention can be implanted at the site to be treated. Thereby, theformulations of the present invention, when including a localanesthetic, may be used in the control of post-operative pain.

The local anesthetic is incorporated into the polymer or othersustained-release formulation in a percent loading between 0.1% and 90%or more, by weight, preferably between 5% and 80%, or more, by weightand more preferably between 65 and 80%, or more, by weight. In an evenmore preferred embodiment, the local anesthetic is loaded at about 75%by weight.

It is possible to tailor a system to deliver a specified loading andsubsequent maintenance dose by manipulating the percent drugincorporated in the polymer and the shape of the matrix or formulation,in addition to the form of local anesthetic (e.g., free base versussalt) and the method of production. The amount of drug released per dayincreases proportionately with the percentage of drug incorporated intothe formulation, e.g., matrix (for example, from 5 to 10 to 20%). In thepreferred embodiment, polymer matrices or other formulations with about75% drug incorporated are utilized, although it is possible toincorporate substantially more drug, depending on the drug, the methodused for making and loading the device, and the polymer.

When the augmenting agent is included in the sustained releasesubstrates (e.g., microparticles) comprising local anesthetic, it hasbeen found that useful loadings of augmenting agent are from about0.001% to about 30% by weight of the substrate or preferably from about0.01% to about 5% by weight of the substrate. When the augmenting agentis included in sustained release substrates (e.g., microparticles)without local anesthetic, it has been found that useful loadings ofaugmenting agent are from about 0.001% to about 90%, or more, by weightof the substrate, or preferably from about 0.001% to about 30% by weightof the substrate or more preferably from about 0.01% to about 5% byweight of the substrate.

When the augmenting agent is included as part of the (aqueous) injectionmedium, the augmenting agent may be present in a weight percent relativeto the local anesthetic varying from about 0.01% to about 15%.

The dosage of the sustained release microsphere formulations isdependent upon the kind and amount of the drug to be administered, therecipient animal, and the objectives of the treatment. For example, whenthe local anesthetic included in the microspheres of the presentinvention is bupivacaine, the formulation may include, e.g., from about0.5 to about 2 mg/kg body weight. The effective dose of bupivacaine, oran amount of another local anesthetic sufficient to provide proportionalpotency, can range from about 1 to 50 mg of bupivacaine injected orinserted at each site where the release of a local anesthetic agent isdesired. In certain preferred embodiments, the dose of bupivacaine inthe sustained release dosage form of the invention is sufficient toprovide a sustained release of about 1 to 4 mg per day at the releasesite for at least 1 to 4 days. Since the formulations of the presentinvention are sustained release, it is contemplated that formulationsmay include much more than usual immediate release doses, e.g., as muchas 120 mg/kg bupivacaine or more.

In certain preferred embodiments, the sustained release substrate (e.g.,microparticles) comprising local anesthetic and/or augmenting agentprovides from about 10 to about 60 percent release of drug, e.g., localanesthetic after 24 hours, from about 20 to about 80 percent releaseafter 48 hours and from about 40 to about 100 percent release after 72hours. In such embodiments, it is preferred that the sustained releaseformulation provide anesthesia and/or local numbness and/or pain reliefat the desired site for about 3-5 days. More preferably, the sustainedrelease substrate comprising local anesthetic provides from about 25 toabout 40 percent release of local anesthetic after 24 hours, from about40 to about 50 percent release after 24 hours and from about 45 to about55 percent release after 72 hours and 80 to 100 percent cumulativerelease is provided after about 280 hours. In such embodiments, it ispreferred that the sustained release formulation provide anesthesiaand/or local numbness and/or pain relief at the desired site for about3-5 days.

In order to obtain a local anesthetic effect in-vivo when combined withthe augmenting agent as described herein of at least about 40 hours theaugmenting agent is placed into approximately the same site in a patient(e.g., human or veterinary) before, simultaneously with, or after theplacement of a local anesthetic at that site. The presence of augmentingagent in the sustained release formulation does not significantly affectthe in-vitro release rates of local anesthetic.

In a preferred embodiment the local anesthetic effect is prolonged bythe use of an augmenting agent by at least about 15%, e.g., from about15% to about 1400% or more preferably from about 300% to about 1000% ormore and more preferably from about 300% to about 500%, or more of theduration of the local anesthetic effect that is obtained from the sameformulation without benefit of an augmenting agent. The duration of thelocal anesthetic effect prolonged by an augmenting agent ranges fromabout 30 minutes to about 150 hours, or more, and preferably from 1 hourto about 1 to about 24 hours or more, and more preferably from about 1hour to about 12 hours, or more.

The rate of release of local anesthetic agent or other drugsincorporated into the formulation will also depend on the solubilityproperties of the local anesthetic or drug. The greater the solubilityin water, the more rapid the rate of release in tissue, all otherparameters being unchanged. For example, those local anesthetic agentshaving pH dependent solubility will be released more rapidly at theoptimum pH for those compounds. Thus, the formulation may be optimizedfor the desired local anesthetic release rate by selecting localanesthetic agents having a desired water solubility in tissue, e.g., attissue pH. Thus, a local anesthetic agent that is more soluble at acidpH will have a faster release rate in a relatively acidic (e.g., pH lessthan about 7.2) tissue. For example, in one embodiment, the formulationwill have released, in vitro, at least 70 percent of a local anestheticat 48 hours at about pH 6 and will have released at least 40 percent ofa local anesthetic at a pH ranging from about 7.4 to about 8, at 48hours. Other combinations are pH independent in their release.

The examples demonstrate that the above-described augmenting agentsprolong the duration of local anesthesia in-vivo and do notsignificantly alter the time course of release of bupivacaine in-vitro.

Applications

Potential applications include any condition for which intra articularsustained release of one or more of the active agents enumeratedhereinabove is desirable. In a preferred embodiment, potentialapplications include any condition for which localized anesthesia and/oranti inflammatory activity is desirable. Preferably, the formulationsaccording to the invention are inserted, injected, infiltrated orinfused into an articular joint in need of local anesthesia, e.g.,prevention or reduction of pain sensation. Thus, painful joints can betreated with local anesthetic having prolonged effect. Conditions to betreated by the formulations according to the invention include lowerback pain, neck pain, including, e.g., whiplash pain in the affectedjoint or joints, e.g., the zygopohyseal joints, pain in the joints ofthe extremities such as knee and elbow joints caused by disease and/orby trauma.

In a preferred embodiment, the methods of the invention are particularlysuited for the treatment of arthritic joint disease, e.g., rheumatoidarthritis where a combination of local anesthetic and aglucocorticosteroid antiinflammatory agent provides both immediate andprolonged local anesthetic activity, as well as the additionalantiinflammatory activity when the glucocorticosteroid is administeredin an amount effective for antiinflammatory activity.

In an especially preferred embodiment, the formulation according to theinvention is in the form of a plurality of sustained releasemicroparticles also referred to herein as extended duration localanesthetic (EDLA).

Of course, the aforementioned applications of the methods of theinvention are merely mentioned as examples, and additional applicationsfor both human and veterinary practice will be immediately apparent tothe artisan.

The formulations of the invention are also suitable for administrationin all body spaces/cavities, including but not limited to pleura,peritoneum, cranium, mediastinum, pericardium, bursae or bursal,epidural, intrathecal, intraocular, etc.

The uses of the formulations of the invention includes both localanesthesia for the relief of pain and motor symptoms as well as localanesthesia for other medical purposes. The formulations and methodsaccording to the invention can be used to provide two to five dayintercostal blockade for thoracotomy, or longer term intercostalblockade for thoracic post-therapeutic neuralgia, lumbar sympatheticblockade for reflex sympathetic dystrophy, or three-dayilioinguinal/iliohypogastric blockade for hernia repair. Other potentialapplications include obstetrical or gynecological procedures. Yetfurther potential applications include providing localized temporarysympathectomy, e.g., blockade of sympathetic or parasympathetic gangliato treat a variety of autonomic diseases, including circulatorydysfunction or cardiac dysrhythmias. The formulations may also be usedto treat trigeminal neuralgia and other diseases of the cranial nervesas well as to provide a temporary nerve block to treat localized musclespasm and treatment of retrobulbar conditions, e.g., eye pain. Otheruses include intra-operative administration in order to reduce painduring and after the operative procedure, especially for plastic surgeryprocedures where prolonged local anesthesia will enhance the outcome.These systems can also be used for the management of various forms ofpersistent pain, such as postoperative pain, sympathetically maintainedpain, or certain forms of chronic pain such as the pain associated withmany types of cancer. These systems may also be used for blockade ofnociceptive pathways (afferent and efferent) in patients with acutepancreatitis, ileus, or other visceral disorders. These are merelyexamples, and additional uses for both human and veterinary practice areimmediately apparent to one skilled in the art.

Methods of Administration

In a preferred method of administration an EDLA dosage form, e.g.,microparticles such as microspheres or microcapsules, are administeredby injection into a site where local anesthetic agent is to be released.Microspheres may be injected through a syringe or a trochar. Pellets,slabs or solid formulations shaped to fit particular locations, e.g.,articular joints, may be surgically placed into a site where release oforal anesthetic agent is desired. Sustained release gels, pastes orsuspensions, including gels, pastes or suspension containingmicroparticles, may also be administered topically to a skin or mucosalsurface of the body to obtain topical, localized anesthesia. Fortreatment of joint pain of the back or neck the EDLA may be administeredby intra articular injection into one or more facet joints.

As described below, microspheres according to the invention can beadministered alone or in combination with a solution including aglucocorticoid or non-glucocorticosteroid augmenting agent in an amounteffective to prolong the duration of local anesthesia. Alternatively,the microspheres include an amount of a non-glucocorticosteroid augmentagent effective to prolong the duration of local anesthesia.

In another alternative, one or more augmenting agents can beadministered before, simultaneously with or after administration of thesustained release local anesthetic, wherein the augmenting agent isformulated into a separate microsphere formulation for sustainedrelease. The controlled release rate for the augmenting agents may bethe same as or different than the controlled release rate for the localanesthetic. The separate microsphere can be administered in a singleinjection, i.e., in a single injection vehicle, or in separateinjections simultaneously or at different times. In a furtherembodiment, it has been found that additional dose of augmenting agentmay also be administered as an injectable solution, in an injectablecarrier or in a sustained release carrier to the nerve to be blockadedafter the sustained release local anesthesia has worn off, in order toreactivate the initial local anesthesia without the co-administration ofadditional local anesthetic.

The microspheres may be prepared from PLGA polymers ranging from, forexample, PLGA in a ratio of 50/50, 65/35 or 75/25. An optimumcomposition has been determined to be PLGA 65/35. The microspheres,formulated with, e.g., PLGA 65/35 microspheres are administered in adose ranging from, for example, 2 through 450 mg of microspheres 75%(w/w) loaded with a local anesthetic such as bupivacaine, per kg of thepatient to be treated. In a preferred embodiment the dose ranges from 5through 450 mg/kg. In a more preferred embodiment the dose ranges fromabout 10 to about 150 mg/kg with PLGA 65/35. Certainly, the artisan willappreciate the fact that the dose ranges mentioned above are based onthe potency of bupivacaine, and that exact effective dosages will varywith the particular relative potency and pharmacokinetics of each localanesthetic and will be able to readily adjust the dose according to thedegree of blockade experienced by the patient.

The use of the above-described augmenting agents before, simultaneouslywith or after administration of a sustained release local anesthesia,results in prolonged anesthesia.

A suspension of microspheres prepared in a form suitable for intraarticular injection can be injected into a joint using methods wellknown to the art. For most body spaces, the use of a needle or “skinnyneedle” is acceptable. The chosen needle is one that is small in bore(large) gauge as possible, and as long as necessary. Commonly, for ajoint, epidural, intraperitoneal, intrapleural or bursae, 22-28 gauge,1-2 inch is used. For the microparticles used in the present invention,one should allow for increased bore size (e.g., to 18 gauge). This alsoallows for the puncturing needle to be removable, being encased in aplastic infusion catheter. For a few procedures, “skinny needles” areused. Such needles have the same bores but are longer, and hence look“skinny”. For locations such as intrapericardial, the gauges for theskinny needle are the same, but the needles can be up to 3-4 incheslong. For epidural, and other locations, there is a metal puncturingneedle of the same gauges and up to 3 inches long, often encased in aplastic catheter, through which another catheter, fromm 22-28 gauge, andup to 6-12 inches long, can be inserted into the space.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following non-limiting examples illustrate the preparation of theformulations according to the invention and the effects of localanesthetic and augmenting agents alone and in combination.

EXAMPLES 1-3 Solvent Extraction Process

In Examples 1-3, bupivacaine microspheres are prepared by dissolving thebupivacaine base and the polymer in ethyl acetate. The polymer is 50:50poly (D,L) lactic co-glycolic acid which has a mole percent compositionof 50% lactide and 50% glycolide. This dispersed phase is then added toa solution of polyvinyl alcohol (PVA) in water (the continuous phase)with stirring. The resulting emulsion is monitored for droplet size,which is in turn controlled by the rate of stirring. The emulsion isthen added to water to extract the solvent and to harden themicrospheres. The mixture is then filtered and the microspheres aredried under vacuum at room temperature. The desired particle sizefraction is then collected by sieving.

Each of Examples 1-3 are prepared such that the microspheres have arelatively high drug content. In Example 1, the theoretical drug contentis about 60%, and the size of the microspheres range from about 45 toabout 90 microns. In Example 2, the theoretical drug content is about61%, and the range in the size of the microspheres is from about 45 toabout 63 microns. In Example 3, the theoretical drug content is about65%, and the range in particle size of the microspheres is from about 45to about 63 microns.

The microspheres of Examples 1-3 are then suspended in a suitable mediafor injection, in this case water. Thereafter, the microspheres aresubjected to in-vitro dissolution testing. An automated dissolution testmethod is utilized using the USP/NF Paddle Method II. The dissolutionmedium is 900 ml of Tris buffer with 0.05% sodium dodecyl sulfate at pH7.4 at 37° C. with a stirring speed of about 50 RPM. The surfactant isadded in order to prevent the microspheres from floating on the surfaceof the dissolution medium. Further information concerning theseformulations is presented in Table 1 below.

TABLE 1 M-Sphere MW of Size Theoretical Actual 50:50 In vitro ReleaseFormulation Range % Drug % Drug dl-PLGA 24 hrs 72 hrs Ex. 1 45-90μ 62%47% — 28% 68% Ex. 2 45-63μ 61% 56% 50K 52% 91% Ex. 3 45-63μ 65% 59% 50K22% 46%

From the results set forth in Table 1, no correlation between drugcontent and release rate can be readily made.

It was expected that the formulation of Example 3 would release drugfaster than that of Example 1 because of a higher drug content. However,the in-vitro release for Example 3 was slower than expected. It ishypothesized that this is due to the glass transition temperature of thepolymer being lowered (below about 37° C.) by the high drug content.This situation may or may not be translated into in-vivo results.

EXAMPLES 4-9 Spray-dried

In Examples 4-9, the bupivacaine base and the polymer utilized inExamples 1-3 are once again dissolved in ethyl acetate, but this timethe microspheres are obtained by spray-drying the solution. Example 4utilizes a relatively high drug content, whereas Example 5 utilizes arelatively low drug content. In Examples 7-9, microspheres having asubstantially similar drug content to Examples 4-5 are prepared usingthe solvent extraction technique utilized in Examples 1-3. Details ofthe formulations are presented in Table 2 below.

TABLE 2 Drug Content Formulation (Theoretical) Yield Process Ex. 4 49%55% Spray-Dried Ex. 5 29% 64% Spray-Dried Ex. 6 45% — Spray-Dried Ex. 747% 62% Solvent Extraction Ex. 8 28% 74% Solvent Extraction Ex. 9 60%60% Solvent Extraction

With regard to Example 9, the actual percentage of bupivacaine base inthe microspheres is 51%, the molecular weight of the 50:50 dl-PLGApolymer is 18,000, the microspheres were about 45-63 microns, andin-vitro dissolution conducted as in Examples 1-3 showed that 61% of thebupivacaine was released in 22 hours.

The microspheres of Examples 6 and 9 are suspended in a suitableinjection medium (e.g., water) and then subjected to in-vitrodissolution testing via the procedures set forth in Examples 1-3. Thein-vitro dissolution results are determined for 22 hours.

The in-vitro dissolutions of Examples 4-5 and 7-8 are determined as perthe Examples above, and compared to the dissolution of the bupivacainefree base and the bupivacaine hydrochloride salt forms. When compared topure bupivacaine base, each of Examples 4-5 and 7-8 showed a distinctretarding effect in their dissolution profile. Furthermore, all fourexamples of the invention displayed a small initial burst of drugrelease which was more pronounced in the microspheres prepared by thespray-dried process as compared to the examples prepared by the solventextraction process.

Scanning electron micrographs of the microspheres for the formulationsprepared by the solvent extraction and by the spray-dried technique arethen compared. The spray-dried process yields microspheres which aresmaller than with the solvent extraction process.

EXAMPLE 10 Local Anesthesia Induced by Sustained Release Microspheres isProlonged by Co-Administration of Dextran Augmenting Agent in theInjection Solution

Microspheres are prepared which contain 75% bupivacaine, by weight. Theduration of local anesthesia induced by the bupivacaine-loadedmicrospheres, prepared from PLGA 65:35, with and without theco-administration of an augmenting agent, is tested in a rat sciaticnerve model for localized local anesthesia. In this procedure, groups ofrats are selected that demonstrate normal behavior in a leg withdrawallatency test at least a week prior to the experimental procedure. Thelatency test determines the time, in seconds, before a rat withdraws itshindpaw from a hotplate set to a safe but uncomfortable temperature (56°C.).

Selected rats are injected with a solution containing a suspension ofbupivacaine-loaded microspheres plus co-administered augmenting agent onone side and injected with a control on the contralateral side so thateach rat serves as its own control. Each injection is adjacent to thesciatic nerve. The controls are bupivacaine-loaded microspheres withoutco-administered augmenting agent and microspheres without anybupivacaine.

A. Sensory Testing

As previously discussed, the degree of sensory local anesthesia ismeasured by determining the time or latency, in seconds, before each ratwithdraws its hindpaw from a hotplate set to a safe but uncomfortabletemperature. Maximum sciatic nerve sensory blockade is defined as havinga latency of about 12 seconds or higher.

B. Motor Testing

The degree of motor blockade is measured by scoring the appearance ofthe affected foot for the signs of loss of motor tone. The assessment isconducted as follows using a 4-point scale based on visual observation:(1) normal appearance, (2) intact dorsiflexion of foot with an impairedability to splay toes when elevated by the tail, (3) toes and footremained plantar flexed with no splaying ability, and (4) loss ofdorsiflexion, flexion of toes, and impairment of gait.

C. Experimental Protocol Twenty-four rats each receive an injection ofbupivacaine-loaded sustained release microspheres into the left or rightside, co-administered with a dextran containing injection solution. Thecontralateral side receive either bupivacaine-loaded microspheres at thesame dose, or unloaded microspheres co-administered with a dextraninjection solution.

Sensory hot plate latency is measured from the time of the injectionsuntil the latency declined to under 2 seconds.

Motor blockade is scored until the hind paws of motor blockades ratsreturned to a normal appearance.

The dose of bupivacaine contained in each sciatic nerve injection rangesfrom 5 to 450 mg/kg of rat or about 1.5 to 50 mg at each injection site.

The tested dextrans has a molecular weight ranging from 20 kDa through200 kDa. The injection solution containing dextran is buffered to a pHranging from 7.0 to 8.3.

D. Results

On the sides receiving co-administered dextran augmenting agent show asignificantly longer duration of sensory block and significantlyincreased duration of motor block than do the sides receivingsustained-release bupivacaine-loaded microspheres withoutco-administered dextran. Unloaded microspheres with dextran aloneproduce no sensory blockade.

EXAMPLE 11 Local Anesthesia Induced by Sustained Release Microspheres isProlonged by Co-Administration of Alkalinizing Agents in the InjectionSolution

Preparation of microspheres and testing procedures are as describedabove. In this experiment it is shown that co-administration ofalkalinizing agents in the injection solution serve to significantlyprolong the duration of local anesthesia induced by the injection ofsustained release bupivacaine-loaded microspheres adjacent to ratsciatic nerve.

A. Experimental Protocol

Twenty-four rats each receive an injection of bupivacaine-loadedsustained release microspheres into the left or right side, adjacent tothe sciatic nerve, in carbonate-buffered injection solution. Thecontralateral side receives either bupivacaine-loaded microspheres atthe same dose at pH 7.4, or unloaded microspheres with the sameinjection buffer as the treatment side. The pH of the experimentalinjection solution ranges from pH 7.0 through pH 8.3.

B. Results

The degree of sensory and motor local anesthesia show a significantincrease in duration proportional to the alkalinity of thecarbonate-buffered injection solution, with the optimum results obtainedas the pH approached 8.

EXAMPLE 12 Local Anesthesia Induced by Sustained Release Microsphereswas Prolonged by Co-Administration of Agents with DiversePharmacological Activity

In this example, a large number of pharmaceutical agents were tested foractivity in augmenting the duration of local anesthetic activity.Bupivacaine-containing microspheres at about 75% loading, by weight,were injected perineurally into rat sciatic nerve at a dose of 150 mg/kg(weight microspheres/weight rat) to dose of approximately 50 mg/nerve.For the injections, needle placement adjacent to the target nerve wasoptimized by intermittent electrical e stimulation of the target nerve(via the injection needle) with low amplitude current to produce limbflexion. For the injections, the microspheres were suspended in acarrier vehicle suitable for injection. While any pharmaceuticallyacceptable carrier vehicle is acceptable, for these experiments thecarrier vehicle was 1% sodium carboxymethylcellulose and 1% Tween 80 inwater.

Compounds to be tested were co-injected with bupivacaine containingmicrospheres (i.e., mixed as additives into the carrier vehicle) in arange of concentrations. Results are expressed as percent increase induration relative to non-augmented durations that were obtained in thesame animal model.

The duration of anesthesia was measured by the hotplate foot withdrawalmethod described above in Example 10 and refers to the time necessaryfor the animal to recover from maximal nerve latency (12 sec) to alatency of 7 seconds, approximately 50% recovery. The results aretabulated in Table 3 below as percent of control.

TABLE 3 Efficacy of Additives to LAB As Percent of Control WithoutAdditive Duration Principle % Anesthesia Pharmacological Additive AsPercent Activity of Additive Conc. of Control AdditiveAllotetrahydrocortisone 0.05 100 Steroid, GABA Allotetrahydrocortisone0.5 117 receptor modulator Alphaxalone 0.05 169 Steroid, GABA receptormodulator and anesthetic Alphaxalone 0.5 123 Aminopyridine (4-AP) 0.05 77 Potassium channel Aminopyridine (4-AP) 0.11  92 blockerAminopyridine (4-AP) 1.09 131 Aminopyrine 0.05 146 Analgesic Aminopyrine0.5  62 Benzamil 0.05  83 Sodium channel Benzamil 0.5 154 inhibitorClonidine 0.05 122 Partial 2 adrenergic Clonidine 0.5  71 agonist andvasoconstrictor. Colchicine 0.01 104 Microtubule inhibitor, Colchicine0.1 677, 1308 inhibitor of glucose Colchicine 1.0 277 metabolism inColchicine 10 toxic leukocytes (among Colchicine (Placebo) 0.1  0 otherproperties). Colchicine (no LAB) 10  0 DehydroePiandrosterone 0.05Steroid, GABA DehydroePiandrosterone 0.5 receptor modulator Dextran 3146-144 Osmotic Dextran 6 Anesthesia polysaccharide continued past endof test period Diazepam 0.05 231 Modulates GABA Diazepam 0.5 203receptor Diazoxide 0.05 138 Potassium-ATP Diazoxide 0.5 109 channelagonist 5,5-diphenylhydantoin 0.05 145, 119 Sodium channel5,5-diphenylhydantoin 0.11 152 inhibitor 5,5-diphenylhydantoin 1.09 138Minoxidil 0.05  54 Potassium channel Minoxidil 0.5 218-265 agonistOuabain 0.05 154 Na,K-ATPase inhibitor Ouabain 0.5 178 Spantide 0.05 119Neurokinin antagonist Spantide 0.5 172 Taxol 0.05 188, 138 Microtubuleassembly Taxol 0.11 104 promoter Taxol 0.5  82 Taxol 1.09 108Tetraethylammonium 0.05  95 Potassium channel Tetraethylammonium 0.5 123blocker U-73, 122* 0.05 106 PLC inhibitor U-73, 122* 0.5 115 ValproicAcid 0.05 152 Potassium channel Valproic Acid 0.5 138 opener Vinblastine0.05 158 Microtubule inhibitor Vinblastine 0.11  37 Vinbiastine 1.09 40 *(1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexl]-1-H-pyrrole-2,5-dione)

EXAMPLE 13 Epinephrine as Augmenting Agent

Microspheres containing bupivacaine loaded to about 75 percent by weightwith bupivacaine are prepared, with and without added epinephrine, in apercent loading of about 0.05 percent, by weight, using the methodsdescribed in Examples 1-3 or Examples 4-9, above.

Following the protocol set forth in Example 10, above, selected rats areinjected adjacent to the sciatic nerve with a solution containing asuspension of bupivacaine-loaded microspheres on the right side, and onthe left side with a solution containing a suspension ofbupivacaine-loaded microspheres and also containing 0.05 percentepinephrine.

Sensory and motor testing is conducted according to sections A and B,respectively, of EXAMPLE 10, above. Using the experimental protocol ofsection C of EXAMPLE 10, 24 rats are tested.

On the sides receiving a combination of bupivacaine and epinephrine insustained release microspheres, a significantly longer duration ofsensory block and significantly increased duration of motor block wasobtained than with the sides receiving sustained-releasebupivacaine-loaded microspheres without sustained release epinephrine.

EXAMPLE 14 Amphetamine as Augmenting Agent

In experiments conducted according to EXAMPLE 13, above, amphetamine issubstituted for epinephrine, with the same concentrations of each agent.On the sides receiving a combination of bupivacaine and amphetaminecontaining controlled release microspheres, a significantly longerduration of sensory block and significantly increased duration of motorblock was obtained than with the sides receiving sustained-releasebupivacaine-loaded microspheres without sustained release amphetamine.

EXAMPLE 15 Ephedrine as Augmenting Agent

Microspheres containing bupivacaine loaded to about 75 percent by weightwith bupivacaine are prepared, in a percent loading of about 0.05percent, by weight, using the methods described in EXAMPLES 1-3 orEXAMPLES 4-9, above. In addition, microspheres containing addedephedrine, in a percent loadings of 0.001 percent, 0.05 percent and 1percent, without bupivacaine, are also prepared according to EXAMPLES1-3 or EXAMPLES 4-9, above.

Following the protocol set forth in EXAMPLE 10, above, selected rats areinjected adjacent to the sciatic nerve with a solution containing asuspension of bupivacaine-loaded microspheres on the right side, and onthe left side with a solution containing a suspension ofbupivacaine-loaded microspheres and also containing ephedrine containingmicrospheres in each dose level.

Sensory and motor testing is conducted according to sections A and B,respectively, of EXAMPLE 10, above. Using the experimental protocol ofsection C of EXAMPLE 10, 24 rats are tested for each of the threeephedrine dose levels by injecting ephedrine-containing microspheres(same number of microspheres per rat, adjusted for animal weight) atabout the same time as the bupivacaine-containing microspheres areadministered. On the sides receiving a combination of bupivacainemicrospheres and epedrine microspheres, a significantly longer durationof sensory block and significantly increased duration of motor block wasobtained than with the sides receiving sustained-releasebupivacaine-loaded microspheres without sustained release ephedrine foreach dose level, with the effect showing a dose-response curve accordingto concentration.

EXAMPLE 16 In-vivo Injection into Joints

As can be appreciated, a substantial range of pharmaceutical agents iscapable of augmenting the duration of local anesthetic activity. Inaddition, these compounds were tested as additives in the vehiclesuspending the microspheres. Including an augmenting agent into thesustained release formulation itself is expected to substantiallyimprove the prolongation of local anesthetic activity by prolonging thepresence of augmenting agent at the anesthetized site.

EDLA (Extended Duration Local Anesthetic), as the term is used in thisexample, is a formulation of bupivacaine and dexamethasone in a matrixof poly(lactide:glycolide) 65:35 microspheres, which releases thebupivacaine and dexamethasone over a period of several days. The polymeris also biodegradable, and remnants may last for several weeks tomonths. The formulation used in this study consisted of about 72%bupivacaine and 0.04% dexamethasone by mass, the microspheres ranging insize from about 25 μm-125 μm, with a mass median diameter of just over100 μm.

Local anesthetics have been previously injected into joint spaces torelieve pain, with mixed results. The present formulation has beendemonstrated to provide anesthesia having a duration of several days ina number of animal species. However, in order to confirm that the EDLAin the form of microparticles does not cause mechanical damage whenadministered to joint spaces that are freely exercising, the followingexperiments were conducted.

Method of Manufacture

50 mg of poly(lactide:glycolide) (“PLGA”) 65:35 (High molecular weight)and 150 mg of bupivacaine free base (obtained from Purdue-Frederick)were dissolved in 0.1 ml of a solution of 5 mg of dexamethasone in 5 mlsin CH₂Cl₂ and 0.9 mls of CH₂Cl₂. 1 ml of 0.3% polyvinyl alcohol (PVA) in100 mM Tris buffer at pH 8.5 was added and the mixture vortexed 3 timesfor 15 seconds each time. The mixture was poured into 100 mls of 0.1%PVA in 100 mM Tris buffer. The microspheres were examined using thelight microscope and the size distribution was determined, using acoulter counter, to be between 10 and 110 microns. The CH₂Cl₂ wasremoved by heating the sample to 31° C. using a rotary evaporator atfull vacuum for 15 minutes. The suspension of microspheres in 0.1% PVAwas filtered through 140, 60, and 20μ metal sleeves (Neward Wire ClothCo.). Then the microspheres were frozen in liquid nitrogen andlyophilized overnight.

The EDLA microspheres composed of 72% bupivacaine, 0.04% dexamethasone,in poly(lactide:glycolide) (65:35) microspheres, mass median diameter ofabout 110 μm, were suspended in a vehicle of 0.5% sodiumcarboxymethylcellulose and 0.1% Tween 80 in water. All percentages arereported as weight percent unless otherwise specified.

Animals

The experiment was performed using three elderly male baboons that hadpreviously been used in abdominal surgery studies, and which werescheduled to be sacrificed.

Protocol

The objective of the experiment was to inject EDLA microparticles intothe knee joints of adult baboons and measure plasma concentrations ofbupivacaine for several days, observe the movements of the animals forseveral days for induced lameness, physically examine the joints weeklyfor evidence of inflammation, and necropsy the animals and examine thejoints grossly and histologically for lesions. Two animals received aninjection of EDLA microspheres in one knee and vehicle in the other,while one animal received an injection of vehicle in one knee and noinjection in the other knee (Table 4). The protocol was conducted in thefollowing sequence.

Week 1, the animals were on a sham tether.

Week 2, the animals were on a tether.

Week 3, the knee joints were X-rayed and EDLA microspheres (bupivacaineand dexamethasone) were injected intra articularly.

During weeks 4, 5 and 6 serum drug levels, daily observations of walkingwere taken and physical examinations of the joints were conductedweekly. Final X-rays and necropsy was conducted at day 21.

TABLE 4 Administration of Test Substances Animal Joint space Testsubstance A Left knee EDLA, 70 mg in 1 ml Right knee Vehicle, 1 ml BLeft knee Vehicle, 1 ml Right knee EDLA, 70 mg in 1 ml of vehicle C Leftknee No injection Right knee Vehicle, 1 ml

The animals were tethered so that frequent blood samples could be drawnduring the first week after injection to measure plasma bupivacaine.Radiographs were taken prior to the injection of the test article andthree weeks after injection (prior to necropsy) for evidence of lesions.The radiograph (X-rays) were confirmed by pathology studies, includinggross and histologic evaluations.

B. Results

1. In-life Observations

No evidence of inflammation, tenderness or altered range of motion onweekly physical examination of knees, in any animal.

2. Plasma Bupivacaine

FIG. 2 is a graph which depicts the presence of plasma bupivacaine afterthe administration of the EDLA bupivacaine microspheres.

As shown in FIG. 2, Animal A, who was injected with 70 mg of the EDLA inthe left knee, had over 75 ng/ml plasma bupivacaine during day 1.Thereafter the level of bupivacaine decreased to approximately 60 ng/mlafter day 2, and below 25 ng/ml after day 3, until the level reached 0ng/ml after day 5.

Animal B, who was injected with 70 mg of the EDLA in the right knee hadover 50 ng/ml plasma bupivacaine after day 1. Thereafter the level ofbupivacaine decreased to 25 ng/ml after day 2 until the level reached 0ng/ml after day 4.

FIG. 2 confirms that Animal C, who was not injected with the EDLA, hadno plasma bupivacaine during the days of the study.

3. Gross Observations on Necropsy

Joints were examined for swelling, warmth, and discoloration. All werenegative. Range of motion and ease of motion were evaluated and all werejudged normal. Joint capsule fluid was examined for transparency, color,and cells. All were negative. The joint capsule was examined forswelling and thickening, and for discoloration. All were negative. Thecartilaginous surfaces of the medial femur, lateral femur, medial tibia,lateral tibia, medial meniscus, and lateral meniscus were examined forroughness. All were judged normal.

4. Histopathologic Observations

The histopathological evaluations were conducted in a blinded manner.

Gross evaluation criteria were as follows.

Motion, mobility.

Inflammation was scored on a scale of 0-3 based on the observation ofswelling, temperature and color.

Capsule fluid was scored on a scale of 0-3 based on transparency, bloodyand purulent.

Joint capsule was scored on a scale of 0-3 based on swelling and color.

Cartilaginous surfaces were scored on a scale of 0-3, each, at themedical femur, lateral femur, media tibia, lateral tibia, medialmeniscus, lateral meniscus.

Results

All specimens were graded as “0”.

Animal A: (EDLA, left knee): Giant cell formation around foreignmaterial was evident in the synovial membrane of the left knee, withminimal lymphocyte infiltration around the giant cells. Cartilaginoussurfaces were normal. Cartilage and synovial membrane was normal in theright knee. Diagnosis was listed:

Granulomatous arthritis, minimal, left knee.

Animal B: (EDLA, right knee): Giant cell formation around foreignmaterial was evident in the synovial membrane of the right knee, withminimal lymphocyte infiltration around the giant cells. Cartilaginoussurfaces were normal. Cartilage and synovial membrane was normal in theleft knee. Diagnosis was listed:

Granulomatous arthritis, minimal, right knee.

Animal C (diluent, right knee): The cartilaginous surfaces and synovialmembranes were normal in both knees.

C. Conclusions

Injection of EDLA microspheres into the knee joints of normal baboonsresulted in no damage to articulating surfaces when assessed after threeweeks. EDLA particles were trapped in synovial membrane, with minimalforeign body reactions as a consequence. This type of reaction has beenobserved to EDLA and other microsphere formulations in most otherstudies. The incorporation of dexamethasone in EDLA microspheres resultsin an attenuated response; further increase in glucocorticosteroidconcentration in EDLA microspheres may result in even less inflammation,and may itself provide a therapeutic benefit in osteoarthritic joints.

Thus, the local tissue concentration of bupivacaine maintained by theEDLA microsphere formulation mirrors the observation that the localanesthesia produced by EDLA microspheres provides rapid onset andprolonged duration of action.

EXAMPLE 17 In-vivo Intraperitoneal Administration

In Example 17, a study was undertaken to examine intraperitonealadministration to rats. The goal of this exercise was to administer EDLAinto another “cavity,” the intraperitoneal cavity. The biological effectstudied was inhibition of gastrointestinal motility as reflected byincreased transit time through the small intestine. EDLA (low molecularweight polymer) was utilized in this study, and was prepared by formingan oil-in-water emulsion from an aqueous solution containing asurfactant (process water) and an organic solvent (oil) solutioncontaining drug and polymer. Following emulsification, the solvent wasremoved in an aqueous quench allowing the microspheres to harden.Details are as follows:

Materials

Process water (aqueous phase) was prepared as follows: A 1% stocksolution of polyvinylalcohol (PVA) was prepared by the addition of 30 gPVA (Spectrum) to 3.0 L of deionized water and heated while mixing to65-70° C. until dissolved. The PVA solution was cooled to ambienttemperature and q.s. to 3.0 L. Next, 375 ml of the stock PVA solutionwas diluted with 1125 ml of deionized water. Finally, 90 ml (80.1 g) ofethyl acetate NF (Fisher) was stirred into the process water prior toforming the emulsion.

The polymer/drug solution (organic phase) was prepared as follows: 5.6 gof Medisorb 65:35DL PLGA (inherent viscosity=0.34 dl/g) was dissolved in150 ml (133.5 g) of ethyl acetate NF under ambient conditions. Next,0.011 g dexamethasone (Upjohn) was added. Then, 14.4 g of bupivacainebase (Orgamol) was added to the polymer solution and sonicated untildissolved. Finally, the organic phase was filtered through a 0.22 μmPTFE filter. The quench solution consisted of 8 L of deionized water atRT.

The organic phase and the aqueous phase were pumped simultaneouslythrough a ½″ diameter by 21 element static mixer (Cole Parmer) to forman emulsion. The organic phase was pumped at a rate of 500 ml/minute andthe aqueous phase at 1000 ml/minute, into the quench solution, which wasbeing stirred mechanically (500 rpm). The quench solution was thenstirred for 1.5 hour, after which the product was passed through 125 and25 μm sieves. The 25-125 μm portion was collected on 10 μm filter paperand dried 4 hours under vaccuum followed by air drying overnight. Theprocess yield was 11.27 g of bupivacaine/dexamethasone-loadedmicrospheres (EDLA).

Method

Charcoal, 10% in gum acacia, 5%, 0.25 ml, was administered to CD-1 miceby gavage. After 20 minutes, animals were euthanized using CO₂. Thegastrointestinal tract was removed, beginning with the stomach,carefully dissecting the mesentery to avoid stretching the smallintestine, with transection at the ileocolic junction. The tract wasmeasured from the jejunopyloric junction to the ileocolic junction.Finally, the distance traveled by the charcoal meal was measured.Gastrointestinal transit was quantitated as the percentage the mealtraveled through the tract compared to the overall length of the tract.

In the trial experiment, a dose of EDLA (low molecular weight polymer),50 mg in 0.3 ml, was administered 4 hours prior to the charcoal meal. Inanimals receiving vehicle, transit was 68±2%, n=10, compared to 43±4%,n=10 in the animals receiving EDLA, t=5.66, df=18, p<0.0001.

EXAMPLE 18 In-vivo Epidural Injection

In Example 18, the methods and procedures of Example 16 are repeated,except that the formulation is instead used for epidural administration.For epidural administration, a catheter is inserted, either for an acuteadministration or indwelling for chronic administration. The dose peradministration should be 10-150 mg equivalent of bupivacaine, which isthe maximum approved for the aqueous formulation. However, by virtue ofthe formulations used in the present disclosure, it has beendemonstrated that such formulations are up to 40 times more safe thansuch approved formulations; therefore, it is possible that the dose ofbupivacaine can be 40 times greater than the 10-150 mg equivalent citedabove. The vehicle is the same used in other applications: sodiumcarboxymethylcellulose —0.05%; polysorbate 80—0.1%; mannitol—50 mM; pH7.4. The EDLA microspheres are diluted so that administration yields thedesired bupivacaine dose in the desired volume (10 mg—greater than 150mg) in 2 ml to 50 ml.

The examples provided above are not meant to be exclusive. Many othervariations of the present invention would be obvious to those skilled inthe art, and are contemplated to be within the scope of the appendedclaims. Numerous publications are cited herein, the disclosures of whichare incorporated herein by reference in their entireties.

What is claimed is:
 1. A method of treating localized pain, comprisingadministering a formulation into a body space selected from the groupconsisting of pleura, peritoneum, cranium, mediastinum, pericardium,bursae, epidural space, intrathecal space, and intraocular space, saidformulation comprising (a) a local anesthetic incorporated in aneffective amount of a biocompatible, biodegradable sustained releasematerial, which prolongs the release of the local anesthetic from theformulation, and (b) a non-toxic augmenting agent in an amount effectiveto prolong the effect of the local anesthetic in-vivo, to treat painarising from said body space.
 2. A method of treating localized pain,comprising administering a formulation into a body space selected fromthe group consisting of pleura, peritoneum, cranium, mediastinum,pericardium, bursae, epidural space, intrathecal space, and intraocularspace, said formulation comprising (a) controlled release microparticlescomprising a local anesthetic and an effective amount of abiocompatible, biodegradable sustained release polymer selected frompolyanhydrides, copolymers of lactic acid and glycolic acid,poly(lactic) acid, poly(glycolic) acid, polyesters, polyorthoesters,proteins, polysaccharides and combinations thereof and (b) a non-toxicaugmenting agent in an amount effective to prolong the effect of thelocal anesthetic in-vivo, said formulation when administered providingpain relief to said body space, said formulation when administeredin-vivo providing local anesthesia or analgesia or numbness or painrelief for at least about 24 hours.
 3. The method of claim 1, whereinthe formulation further comprises a second active agent selected from anenzyme, an anti-infective agent, an antibody, a diagnostic aid, aradio-opaque dye, a magnetic resonance imaging dye, a radiolabeledagent, and combinations thereof.
 4. The method of claim 2, wherein saidbursae is selected from the group consisting of acromial,bicipitoradial, cubitoradial, deltoid, infrapatellar, ishchiadica, andany other bursae subject to pain.
 5. The method of claim 3, wherein atleast a portion of said second active agent is incorporated into saidmicroparticles.
 6. The method of claim 1, wherein the local anestheticis bupivacaine, the augmenting agent is dexamethasone, and the sustainedrelease material is a poly(lactide co-glycolide).
 7. The method of claim2, wherein said formulation when administered in-vivo provides localanesthesia or analgesia or numbness or pain relief for at least about3-5 days.
 8. The method of claim 2, wherein at least a portion of saidaugmenting agent is incorporated into said microparticles.
 9. The methodof claim 2, wherein said augmenting agent is a glucocorticosteriod. 10.The method claim 2, wherein said microparticles comprise localanesthetic in a percent loading between 65 and 80%, and said augmentingagent is a glucocorticosteroid present in a weight percent relative tothe local anesthetic from 0.005% to 15%.
 11. The method of claim 9,wherein said glucocorticosteriod is selected from the group consistingof dexamethasone, cortisone, prednisone, hydrocortisone, beclomethasonedipropionate, betamethasone, flunisolide, methylprednisone,paramethasone, prednisolone, triamcinolone, alclometasone, amcinonide,clobetasol, fludrocortisone, diflorasone diacetate, fluocinoloneacetonide, fluocinonide, fluorometholone, flurandrenolide, halcinonide,medrysone, and mixtures thereof.
 12. The method of claim 2, wherein aplurality of said microparticles are suspended in a pharmaceuticallyacceptable vehicle for injection.
 13. The method of claim 2, whereinsaid local anesthetic is selected from the group consisting ofbupivacaine, ropivacaine, dibucaine, etidocaine, tetracaine, lidocaine,xylocaine, mixtures thereof, and salts thereof.
 14. A method of treatinglocalized pain arising from a body space in a mammal, comprisinginjecting a formulation into a body space selected from the groupconsisting of pleura, peritoneum, cranium, mediastinum, pericardium,bursae, epidural space, intrathecal space, and intraocular space, saidformulation comprising a pharmaceutically acceptable medium containing aplurality of microspheres comprising a local anesthetic and an effectiveamount of a biocompatible, biodegradable controlled release materialcapable of degrading at least fifty percent in less than two yearsfollowing injection of the formulation in vivo, said controlled releasematerial being selected from the group consisting of polyanhydrides,copolymers of lactic acid and glycolic acid, poly(lactic) acid,poly(glycolic) acid, polyesters, polyorthoesters, proteins,polysaccharides and combinations thereof, said formulation furthercomprising a non-toxic augmenting agent which is (i) incorporated intoand/or onto said microspheres; or (ii) incorporated into saidpharmaceutically acceptable medium, or (iii) incorporated into saidmicrospheres and also incorporated into said pharmaceutically acceptablemedium; said microspheres being included in said formulation in anamount sufficient to obtain reversible local anesthesia or analgesia ornumbness or pain relief or anti-inflammatory effect for at least about24 hours when said formulation is injected into said body space.
 15. Themethod of claim 14, wherein said augmenting agent is aglucocorticosteriod.
 16. A method of prolonging the effect of a localanesthetic agent in body spaces, comprising injecting a formulation intoa body space selected from the group consisting of pleura, peritoneum,cranium, mediastinum, pericardium, bursae, epidural space, intrathecalspace, and intraocular space, said formulation comprising apharmaceutically acceptable medium containing a plurality ofmicrospheres comprising a local anesthetic and an effective amount of abiocompatible, biodegradable controlled release material, saidbiocompatible, biodegradable controlled release material capable ofdegrading at least fifty percent in less than two years followinginjection of the formulation in vivo, said controlled release materialbeing selected from the group consisting of polyanhydrides, copolymersof lactic acid and glycolic acid, poly(lactic) acid, poly(glycolic)acid, polyesters, polyorthoesters, proteins, polysaccharides andcombinations thereof, said formulation further comprising a non-toxicaugmenting agent which is (i) incorporated into and/or onto saidmicrospheres; or (ii) incorporated into said pharmaceutically acceptablemedium, or (iii) incorporated into said microspheres and alsoincorporated into said pharmaceutically acceptable medium; saidmicrospheres being included in said formulation in an amount sufficientto obtain reversible local anesthesia or analgesia or numbness or painrelief or anti-inflammatory effect for at least about 24 hours when saidformulation is injected into said body space.
 17. The method of claim16, wherein said local anesthetic is selected from the group consistingof bupivacaine, ropivacaine, dibucaine, etidocaine, tetracaine,lidocaine, xylocaine, mixtures thereof, and salts thereof.
 18. Themethod of claim 16, wherein said formulation when administered in-vivoprovides local anesthesia or analgesia or numbness or pain relief for atleast about 3 to about 5 days.
 19. The method claim 16, wherein saidmicrospheres are microcapsules.
 20. The method of claim 16, wherein thediameter of the microspheres ranges in size from about 5 microns toabout 200 microns.
 21. The method of claim 16, wherein at least aportion of said local anesthetic is incorporated in said microspheres.22. The method of claim 16, wherein said augmenting agent is aglucocorticosteriod.
 23. A method of prolonging the effect of a localanesthetic agent in body spaces, comprising administering a formulationinto a body space selected from the group consisting of pleura,peritoneum, cranium, mediastinum, pericardium, bursae, epidural space,intrathecal space, and intraocular space, said formulation comprising(a) controlled release microparticles comprising a local anesthetic andan effective amount of a biocompatible, biodegradable sustained releasepolymer selected from polyanhydrides, copolymers of lactic acid andglycolic acid, poly(lactic) acid, poly(glycolic) acid, polyesters,polyorthoesters, proteins, polysaccharides and combinations thereof; and(b) a non-toxic augmenting agent in an amount effective to prolong theeffect of the local anesthetic in-vivo, for providing pain relief to abody space selected from the group consisting of pleura, peritoneum,cranium, mediastinum, pericardium, bursae, epidural space, intrathecalspace, and intraocular space, said bursae selected from the groupconsisting of acromial, bicipitoradial, cubitoradial, deltoid,infrapatellar, and ishchiadica, and said formulation when administeredin-vivo provides local anesthesia or analgesia or numbness or painrelief for at least about 24 hours.
 24. The method of claim 23, whereinsaid augmenting agent is a glucocorticosteriod.
 25. A method of treatinglocalized pain, comprising administering a formulation into anintra-articular joint selected from the group consisting of knee, elbow,hip, sternoclavicular, temporomandibular, carpal, tarsal, wrist, ankle,and any other joint subject to pain, said formulation comprising (a) alocal anesthetic incorporated in an effective amount of a biocompatible,biodegradable sustained release material, which prolongs the release ofthe local anesthetic from the formulation, and (b) a non-toxicaugmenting agent in an amount effective to prolong the effect of thelocal anesthetic in-vivo, to treat pain arising from saidintra-articular joint.
 26. The method of claim 25, wherein saidaugmenting agent is a glucocorticosteriod.
 27. A method of treatinglocalized pain, comprising administering a formulation into a spaceselected from the group consisting of a pleural space, mediastinum, andpericardium, said formulation comprising (a) a local anestheticincorporated in an effective amount of a biocompatible, biodegradablesustained release material, which prolongs the release of the localanesthetic from the formulation, and (b) a non-toxic augmenting agent inan amount effective to prolong the effect of the local anestheticin-vivo, to treat pain arising from said space.
 28. A method of treatinglocalized pain, comprising administering a formulation into a spaceselected from the group consisting of a pleural space, mediastinum, andpericardium, said formulation comprising (a) controlled releasemicroparticles comprising a local anesthetic and an effective amount ofa biocompatible, biodegradable sustained release polymer selected frompolyanhydrides, copolymers of lactic acid and glycolic acid,poly(lactic) acid, poly(glycolic) acid, polyesters, polyorthoesters,proteins, polysaccharides and combinations thereof; and (b) a non-toxicaugmenting agent in an amount effective to prolong the effect of thelocal anesthetic in-vivo, said formulation when administered providingpain relief to said space, said formulation when administered in-vivoproviding local anesthesia or analgesia or numbness or pain relief forat least about 24 hours.
 29. A method of treating localized pain arisingfrom a space in a mammal, comprising injecting a formulation into aspace selected from the group consisting of a pleural space,mediastinum, and pericardium, said formulation comprising apharmaceutically acceptable medium containing a plurality ofmicrospheres comprising a local anesthetic and an effective amount of abiocompatible, biodegradable controlled release material capable ofdegrading at least fifty percent in less than two years followinginjection of the formulation in vivo, said controlled release materialbeing selected from the group consisting of polyanhydrides, copolymersof lactic acid and glycolic acid, poly(lactic) acid, poly(glycolic)acid, polyesters, polyorthoesters, proteins, polysaccharides andcombinations thereof, said formulation further comprising a non-toxicaugmenting agent which is (i) incorporated into and/or onto saidmicrospheres; or (ii) incorporated into said pharmaceutically acceptablemedium, or (iii) incorporated into said microspheres and alsoincorporated into said pharmaceutically acceptable medium; saidmicrospheres being included in said formulation in an amount sufficientto obtain reversible local anesthesia or analgesia or numbness or painrelief or anti-inflammatory effect for at least about 24 hours when saidformulation is injected into said space.
 30. A method of prolonging theeffect of a local anesthetic agent in spaces, comprising injecting aformulation into a space selected from the group consisting of a pleuralspace, mediastinum, and pericardium, said formulation comprising apharmaceutically acceptable medium containing a plurality of microspheres comprising a local anesthetic and an effective amount of abiocompatible, biodegradable controlled release material, saidbiocompatible, biodegradable controlled release material capable ofdegrading at least fifty percent in less than two years followinginjection of the formulation in vivo, said controlled release materialbeing selected from the group consisting of polyanhydrides, copolymersof lactic acid and glycolic acid, poly(lactic) acid, poly(glycolic)acid, polyesters, polyorthoesters, proteins, polysaccharides andcombinations thereof, said formulation further comprising a non-toxicaugmenting agent which is (i) incorporated into and/or onto saidmicrospheres; or (ii) incorporated into said pharmaceutically acceptablemedium, or (iii) incorporated into said microspheres and alsoincorporated into said pharmaceutically acceptable medium; saidmicrospheres being included in said formulation in an amount sufficientto obtain reversible local anesthesia or analgesia or numbness or painrelief or anti-inflammatory effect for at least about 24 hours when saidformulation is injected into said space.
 31. A method of prolonging theeffect of a local anesthetic agent in spaces, comprising administering aformulation into a space selected from the group consisting of a pleuralspace, mediastinum, and pericardium, said formulation comprising (a)controlled release microparticles comprising a local anesthetic and aneffective amount of a biocompatible, biodegradable sustained releasepolymer selected from polyanhydrides, copolymers of lactic acid andglycolic acid, poly(lactic) acid, poly(glycolic) acid, polyesters,polyorthoesters, proteins, polysaccharides and combinations thereof; and(b) a non-toxic augmenting agent in an amount effective to prolong theeffect of the local anesthetic in-vivo, for providing pain relief to aspace selected from the group consisting of a pleural space,mediastinum, and pericardium, and said formulation when administeredin-vivo provides local anesthesia or analgesia or numbness or painrelief for at least about 24 hours.